Cell culture techniques 4 - Cryopreservation

Summary

This video from Shamus Biology's animal cell culture series focuses on cryopreservation, a critical process for preserving cells for future research. It explains the importance of cryopreservation in reducing contamination risks and maintaining cell integrity. The script details the steps for preparing cells during the log phase, using cryoprotectants like DMSO to prevent ice crystal formation, and the gradual freezing process to ensure cell viability post-thawing.

Takeaways

• 🧊 Cryopreservation is essential for preserving cell cultures to prevent waste of time and resources after extensive experimentation.
• 🛡️ Cryopreservation reduces the risk of microbial contamination, cross-contamination with other cell lines, and genetic drift or morphological changes due to the cessation of cellular activity at low temperatures.
• 🌡️ Cells should be cryopreserved during the log phase of growth when they are most viable, avoiding the plateau phase where viability decreases.
• 🔬 The process of cryopreservation begins with passaging the cells and ensuring they are in a healthy, viable state before preservation.
• 🌀 After passaging, cells are centrifuged to remove debris and resuspended in a serum-containing media to prepare for cryopreservation.
• 🧪 DMSO (dimethyl sulfoxide) is used as a cryoprotectant to prevent the formation of ice crystals that could damage or kill the cells during freezing.
• 🔬 The exact mechanism of how DMSO protects cells is not fully understood, but it is known to be effective in cryopreservation.
• 💧 The addition of 10% DMSO in Fetal Calf Serum (FCS) is a common method to prepare cells for cryopreservation.
• 🧊 Cells are first frozen at -80°C before being transferred to liquid nitrogen at -196°C to ensure a controlled and slow freezing process.
• ⏱ The gradual decrease in temperature is crucial for maintaining cell integrity and morphology, avoiding rapid changes that could harm the cells.
• 📚 Understanding the different stages of cell culture and growth phases is important for effective cryopreservation and should be reviewed for optimal results.

Q & A

Why is cryopreservation of cells important in cell culture processes?

Cryopreservation is important because it allows the storage of cells for future use, reducing the risk of microbial contamination, cross-contamination with other cell lines, and genetic drift or morphological changes. It ensures that valuable cell properties are preserved for future research.

How does cryopreservation reduce the risk of microbial contamination?

Cryopreservation reduces the risk of microbial contamination because cells are stored at very low temperatures, where microbial activity is significantly reduced or stopped.

What are the benefits of storing cells at low temperatures?

Storing cells at low temperatures reduces the risk of microbial contamination, cross-contamination with other cell lines, genetic drift, and morphological changes. It also preserves the cells' viability for future use.

What is the significance of the log phase in the cell culture growth cycle for cryopreservation?

The log phase is significant because cells are most viable during this phase. Storing cells from the log phase ensures higher viability when they are thawed and used in future research.

What steps are involved in the cryopreservation process after cell passaging?

After cell passaging, the steps involved are: checking for the log phase, resuspending the cells in serum-containing media, centrifugation to remove debris, resuspending cells in 10% DMSO in FCS, transferring the cells to cryovials, freezing at -80°C, and then transferring to liquid nitrogen at -196°C.

Why is DMSO used in the cryopreservation process?

DMSO is used because it acts as a cryoprotectant, helping to prevent the formation of ice crystals inside the cells, which can cause cell death by cracking the cell open.

What is the purpose of freezing cells at -80°C before transferring to liquid nitrogen?

Freezing cells at -80°C before transferring to liquid nitrogen is done to slowly reduce the temperature, preventing rapid changes that could damage the cells. This gradual reduction helps maintain cell integrity.

What is the final temperature used for long-term cell storage in cryopreservation?

The final temperature used for long-term cell storage in cryopreservation is -196°C, typically achieved using liquid nitrogen.

What potential problems does cryopreservation help to avoid?

Cryopreservation helps to avoid microbial contamination, cross-contamination with other cell lines, genetic drift, morphological changes, and the formation of damaging ice crystals within cells.

What are the key properties of cells that cryopreservation helps to maintain?

Cryopreservation helps to maintain the cells' viability, genetic stability, and morphological integrity, ensuring that the cells remain suitable for future research.

Outlines

00:00

🧊 Cryopreservation: Importance and Process

This paragraph discusses the critical stage of cryopreservation in cell culture, emphasizing its necessity for preserving cell properties and experiments for future use. The speaker explains the benefits of cryopreservation, such as reduced risk of microbial contamination, cross-contamination with other cell lines, and prevention of genetic drift and morphological changes. The process involves selecting cells in the log phase of growth for high viability, followed by passaging and centrifugation to remove debris. Cells are then resuspended in a serum-containing medium with 10% DMSO, a cryoprotectant that prevents ice crystal formation which could damage the cells. The paragraph concludes with the initial step of transferring the cell suspension to cryovials for freezing at -80°C.

05:03

🛫 Transition from -80°C to Liquid Nitrogen Storage

The second paragraph continues the discussion on cryopreservation by detailing the transition of cell samples from -80°C to liquid nitrogen storage at -196°C. It highlights the importance of gradual temperature change to avoid altering the cell's morphology, which is crucial for maintaining cell integrity. The process involves an initial freezing step at -80°C before transferring the samples to liquid nitrogen, ensuring a slow and controlled decrease in temperature. This method is essential for the proper preservation of cells, allowing for future research without the risk of damage due to rapid temperature fluctuations.

Keywords

💡Cryopreservation

Cryopreservation is the process of preserving biological cells, tissues, or organs by freezing them at very low temperatures to prevent decay. In the context of the video, cryopreservation is crucial for preserving the cells' viability and integrity for future use. The script emphasizes its importance in reducing risks of contamination, cross-contamination, and genetic drift, as well as maintaining the cells' biochemical pathways in a halted state.

💡Cell Culture

Cell culture refers to the process of growing cells outside their natural environment, typically in a laboratory setting. The video discusses the importance of cryopreservation in the context of cell culture, highlighting the need to preserve cells after they have been studied or manipulated for research purposes. The script mentions the stages of cell culture, including the log phase, which is critical for selecting viable cells for cryopreservation.

💡Log Phase

The log phase, short for logarithmic phase, is a period in the growth cycle of cells where they rapidly divide and grow. The script specifies that cells in the log phase are more than 90% viable, making them ideal candidates for cryopreservation. This phase is contrasted with the plateau phase, where cell growth slows and viability decreases.

💡Viability

Viability in cell culture refers to the ability of cells to live, grow, and reproduce after being preserved and stored. The script stresses the importance of selecting cells with high viability from the log phase for cryopreservation to ensure they will grow properly when thawed and used in future research.

💡Centrifugation

Centrifugation is a technique used to separate components of a mixture by spinning it at high speeds. In the context of the video, centrifugation is used to remove debris and concentrate the cells after passaging, preparing them for cryopreservation. The script describes the process of resuspending the cells in serum-containing media and then centrifuging to pellet the cells.

💡DMSO

DMSO, or dimethyl sulfoxide, is a cryoprotective agent used in cryopreservation to prevent the formation of ice crystals within cells, which could damage or kill them. The script explains that DMSO is added to the cell suspension before freezing to protect the cells during the cryopreservation process, although the exact mechanism is not fully understood.

💡FCS

FCS stands for Fetal Calf Serum, a component of cell culture media that provides nutrients and growth factors necessary for cell growth. In the script, FCS is mentioned as part of the media in which cells are resuspended after centrifugation and before the addition of DMSO for cryopreservation.

💡Cryovial

A cryovial is a small, usually sterile, vial designed to hold samples at cryogenic temperatures. The script describes transferring the cell suspension with DMSO into cryovials as part of the cryopreservation process, which is an essential step before placing the cells into a -80°C freezer and eventually into liquid nitrogen.

💡-80°C Freezer

The -80°C freezer is a type of ultra-low temperature freezer used for the initial stage of cryopreservation. The script mentions that after resuspending the cells in the cryoprotective media, they are transferred to cryovials and frozen at -80°C before being moved to liquid nitrogen for long-term storage.

💡Liquid Nitrogen

Liquid nitrogen is used as a coolant in cryopreservation due to its extremely low temperature of -196°C. The script explains that after the initial freezing at -80°C, the cells are transferred to a liquid nitrogen tank for long-term storage, ensuring the cells remain in a stable, preserved state.

💡Genetic Drift

Genetic drift refers to random changes in the frequency of gene variants (alleles) in a population. In the context of the video, genetic drift is mentioned as a risk that cryopreservation helps to mitigate, as the low temperatures halt cellular activity, preventing changes in the genetic makeup of the cells over time.

Highlights

Cryopreservation is essential for preserving the results and properties of cell cultures for future use.

Failure to preserve cell cultures can lead to a waste of time and money invested in the research.

Cryopreservation reduces the risk of microbial contamination due to the low-temperature storage conditions.

It minimizes the risk of cross-contamination with other cell lines during storage.

Cryopreservation slows or stops biochemical pathways, preventing genetic drift and morphological changes in cells.

Cells should be cryopreserved at a low passage rate to maintain their original properties.

Cells should be in the log phase of growth for optimal viability before cryopreservation.

The plateau phase of cell growth is not suitable for cryopreservation due to reduced cell viability.

Understanding the different stages of cell culture is crucial for effective cryopreservation.

Cells are prepared for cryopreservation by passaging and media exchange to ensure viability.

Centrifugation is used to remove debris and concentrate the cells for cryopreservation.

DMSO is used as a cryoprotectant to prevent the formation of ice crystals that can damage cells.

The mechanism by which DMSO protects cells during cryopreservation is not fully understood but is effective.

Cells are resuspended in a solution containing 10% DMSO in FCS before freezing.

Cryovials are used for the initial freezing process at -80°C to ensure slow and controlled freezing.

Transferring cryopreserved cells to liquid nitrogen at -196°C is the final step for long-term storage.

The gradual transition from -80°C to -196°C is necessary to avoid rapid changes in temperature that can harm cells.

Transcripts

[Music]

welcome back guys welcome to another

session of shos biology

presents animal cell culture series now

in this video we'll be talking about the

stage which is very very important uh

for any cell culture process and it is a

cry of preservation of the cells now we

need to preserve what we have done what

whatever cell we have taken we've done

experiments on it we know some property

of the cells using experiments then we

need to store the cells for the future

so that somebody else once get that cell

know certain properties that that have

been already studied so preservation is

very very important after doing all this

task if we can't preserve it then it

will be a waste of money and time both

the thing so for that c preservation is

the must so two things again two

questions that we want to ask that is

why we require C preservation of the

cells and second is how do we preserve

cells in this uh freezing process so

actually cry preservation of the cells

reduces the risk of microbial

contamination because in Cryo Storage it

can be stored with much less probability

for any contamination than in any other

means of storage second is it reduces

the risk of cross contamination with

other cell lines third is the reduction

of the risk of genetic drift and

morphological changes because in huge

highly cold temperatures there won't be

any cellular activity present inside the

cell so they will kind of just just

thought you know they will just stay as

they are the biochemical Pathways will

be slowed or ultimately it will be

stopped inside the cell so no chances of

cross contamination or any genetic drift

or any morphological changes may occur

there and research conducted using stem

using cells all consented the low

passage rate so for that reason after

certain passage you need to store them

very very much

important so now how do we preserve all

those cells so let's talk about them so

we begin with you know uh passaging of

the cells you know once the cells are

being passaged passage means uh they are

subcultured from the old culture media

to the new culture media once the

process is done we need to check for you

know the log phrase of the growth I

means uh during the log phase only those

cells becomes 90% or above 90% viable

after the lock phase it reaches the

plateau phase of the growth and during

that platty phase of the growth those

sales are not that viable so even if you

store those platy phasic uh sales there

in the future once uh in in future once

we do want to do research on those cells

again we take the cells out from the cry

preserve and put it into the media they

will not grow properly we want some

cells which are having higher viability

and we only get the cells from the log

uh phase of the growth cycle of the

animal cell culture you know if you

don't know the phases of animal cell

culture you you must go and watch the

video from Shamus biology about uh the

different stages of cell culture in

different growth phases of animal cell

culture and then come back

here now after that once you know that

it's it's it's in the lock phase

everything is fine then what we do we

need to passess the sales and P it for

media exchange and passaging and pting

for media exchange is already done after

that you know we resuspend the cells in

serum containing the media and then we

do a centrifugation for pting out all

those uh all those other debris you know

all those other debris are kind of uh

peted and aspirate the super neted so

once we asate the super neted and then

we take the Super nettin and resuspend

that cell because the supernant contains

the cell other debris are wearing the

pet after the centrifugation we just

throw the pet away we take the Super

neted we take it we suspend it in 10%

DMSO in

FCS right DMSO is a cryo preservant that

means it is helped it is helping the

cell to be preserved because you know

the major problem with cryopreservation

and de freezing is forming of uh water

crystals inside uh the cell if it forms

the cell will die because the crystals

will crack the cell open and it will

kill the cell so to prevent that we need

to add certain preservance like DMSO in

this case right the precise mechanism uh

how this DMSO protects the cell is

unknown but we know that DMSO can do

that so it's good for us we place it and

we suspended after that what we do we

transfer this cell to the cryovial right

so

normally we sometimes put it into the

slightly uh small uh small freeze after

the Cal process we freeze atus 80° CI

temperature uh and uh rapid slow

freezing is there in minus 80 that's the

first thing to do after- 80° C we take

it and then we transfer to the liquid

nitrogen tank and that temperature is

minus

196° C so in any way uh it's a very very

general concept that we cannot rapidly

uh transfer the heat we cannot rapidly

change the Heat or change any kind of

climate we need to slowly increase the

Heat or slowly decrease the heat for the

develop M for the proper sequential

growth and without any alteration of

morphology of the cell for that reason

we need to First transfer them to the

cryovial and freeze it at minus 80° C

then we slowly upgrade that to minus

190° C 196° cus temperature in the

liquid nitrogen

tank so that's how it's done