Cell Culture 101 1

Summary

In this educational video, molecular researcher Laura from the University of Toronto introduces the fundamentals of cell culture, specifically focusing on the MCF7 breast cancer cell line. She explains the importance of maintaining a sterile environment using airflow hoods and ultraviolet lights to prevent contamination. Laura demonstrates how to prepare cell culture media with supplements like fetal bovine serum and antibiotics, and how to passage cells using trypsin-EDTA. She also discusses the concept of cell confluence and the process of treating cells with estradiol, highlighting the steps involved in cell culture for scientific research.

Takeaways

• 🔬 Laura is a molecular researcher at the University of Toronto introducing cell culture basics.
• 🌟 Cell culture involves growing eukaryotic cells in vitro for scientific research purposes.
• 🧫 The MCF7 breast cancer cell line is used for demonstration in this tutorial.
• 🔄 Maintaining a sterile environment is crucial in cell culture to prevent contamination.
• 🌬️ Airflow hoods and ultraviolet lights are used to maintain sterility in the cell culture area.
• 💧 Cell culture media, like DMEM, provides essential nutrients for cell survival.
• 💉 Fetal bovine serum is added to the media to supply growth factors for cell growth.
• 🛡️ Antibiotics like penicillin and streptomycin are used to prevent bacterial contamination.
• 🧑‍🔬 The process includes steps like rinsing cells with PBS and using trypsin to detach them from the culture vessel.
• 📊 Cell confluence is an important measure, indicating how densely cells cover the culture plate.
• 🔄 Cells are passaged before reaching 100% confluence to avoid contact inhibition and cell death.
• 🧪 Post-passaging, cells are treated with substances like estradiol for experimental purposes.

Q & A

What is cell culture and why is it important in scientific research?

Cell culture is the growth of eukaryotic cells in vitro for scientific purposes. It is important because it allows researchers to study cell behavior, growth, and response to various treatments in a controlled environment.

Why is maintaining a sterile environment crucial in cell culture?

A sterile environment is crucial to prevent contamination from mold or bacteria, which could invalidate the results and require researchers to start the experiment again.

How do airflow hoods help maintain a sterile working environment in cell culture?

Airflow hoods maintain a sterile environment by generating a current of air that is pulled down into the hood and back through a system of filters, ensuring the air inside the hood is sterile.

What is the role of ultraviolet lights in a cell culture hood?

Ultraviolet lights mounted on the back of the hood irradiate the working surface, further reducing the potential for contamination.

What is the primary function of cell culture media like DMEM?

Cell culture media, such as DMEM, provides a balanced salt solution, pH balance, and essential nutrients necessary for cell survival.

Why is fetal bovine serum added to the cell culture media?

Fetal bovine serum is added because it contains growth factors that stimulate the growth of the breast cancer cell lines being cultured.

What is the purpose of supplementing the media with penicillin and streptomycin?

The supplementation with penicillin and streptomycin acts as a prophylactic measure to prevent any potential bacterial contamination in the cell culture.

How is the cell culture bottle prepared before use in the hood?

The bottle is prepared by spraying it with a 75% ethanol solution to disinfect the surface before placing it in the cell culture hood.

What is the function of trypsin EDTA in cell culture?

Trypsin EDTA is a digestive enzyme that digests external cellular proteins, allowing cells to lift off from the bottom of the culture vessel and be suspended in the media for passaging.

Why are cells passaged before they reach 100% confluence?

Cells are passaged before reaching 100% confluence to prevent contact inhibition, a phenomenon where cells stop dividing and may begin to die when they are too closely packed together.

How does the cell culture incubator contribute to the growth of cells?

The cell culture incubator maintains optimal conditions of 37 degrees Celsius and 5% CO2, simulating the internal environment of the human body, which is conducive to cell growth.

Outlines

00:00

🔬 Introduction to Cell Culture

In this segment, Laura, a molecular researcher at the University of Toronto, introduces the concept of cell culture, which is the growth of eukaryotic cells outside the body for scientific research. She uses the MCF7 breast cancer cell line as an example to demonstrate the process of growing, harvesting, and treating cells. The video emphasizes the importance of maintaining a sterile environment in cell culture to prevent contamination from mold or bacteria, which could invalidate results. To achieve this, Laura uses an airflow hood that creates a barrier between the external environment and the internal working environment by pulling air through filters. Additionally, UV lights are used to irradiate the working surface. The first reagent introduced is cell culture media, specifically DMEM (Dulbecco's Modified Eagle Medium), which provides necessary nutrients and is supplemented with 10% fetal bovine serum for growth factors. A prophylactic administration of penicillin and streptomycin is also used to prevent bacterial contamination. The process of preparing the cell culture media and the use of reagents like PBS (Phosphate-Buffered Saline) and trypsin-EDTA for cell passaging are also covered.

05:02

🌱 Cell Passaging and Confluence

This part of the video script explains the process of cell passaging, which involves removing the existing media, rinsing the cells with PBS, and applying trypsin to detach the cells from the culture vessel. The cells are then diluted in fresh media and transferred to new plates. The concept of cell confluence is introduced, which refers to the population density of cells in a plate. Confluence is measured by the amount of space surrounding the cells, and it's important not to let cells reach 100% confluence to avoid contact inhibition, a phenomenon where cells stop dividing when they are too close together. The video also discusses the treatment of cells with estradiol or vehicle, which involves calculating dilutions and allowing the cells to grow for specific time periods (12, 24, and 48 hours). The cells are then returned to the incubator to adhere to the plate before treatment.

Keywords

💡Cell Culture

Cell culture refers to the process of growing cells outside of their natural environment, typically within a laboratory setting. In the video, cell culture is the central theme, focusing on the growth of eukaryotic cells, specifically the MCF7 breast cancer cell line, for scientific research. The process involves maintaining these cells in a controlled environment to observe and manipulate them for various experimental purposes.

💡Sterile Environment

A sterile environment is a condition where all forms of life and biological contaminants are absent, which is crucial in cell culture to prevent contamination that could compromise the integrity of the experiment. The video emphasizes the importance of maintaining a sterile environment using airflow hoods and ultraviolet lights to ensure that the cell cultures remain uncontaminated.

💡Airflow Hood

An airflow hood is a safety device that provides a barrier between the user and the materials being handled, particularly useful in cell culture to prevent contamination. As described in the video, airflow hoods work by drawing air down into the hood and filtering it, thus creating a sterile workspace. This is essential when handling cell cultures to avoid introducing mold or bacteria into the samples.

💡Cell Culture Media

Cell culture media is a nutrient solution designed to support the growth of cells in vitro. The video mentions the use of DMEM (Dulbecco's Modified Eagle Medium), which provides a balanced salt solution, pH, and essential nutrients for cell survival. Media is often supplemented with other components like fetal bovine serum to enhance cell growth.

💡Fetal Bovine Serum

Fetal bovine serum (FBS) is a component often added to cell culture media to provide growth factors necessary for cell proliferation. In the video, FBS is sourced from the plasma of fetal cows and is used to stimulate the growth of the MCF7 breast cancer cell line, illustrating its critical role in cell culture for promoting cell health and division.

💡Penicillin and Streptomycin

Penicillin and streptomycin are antibiotics used in cell culture to prevent bacterial contamination. The video describes their use as a prophylactic measure at a 1% concentration to ensure the cell cultures remain free from bacterial growth that could interfere with experimental results.

💡Phosphate-Buffered Saline (PBS)

Phosphate-Buffered Saline (PBS) is a saline solution that is buffered to a pH of 7.3, used in cell culture to wash cells and remove any remnants of media. As mentioned in the video, PBS is essential for preparing cells for further manipulation, such as passaging, by ensuring that the cells are clean and free from unwanted debris.

💡Trypsin-EDTA

Trypsin-EDTA is a digestive enzyme solution used in cell culture to detach cells from the surface they are grown on. In the video, trypsin is applied to the MCF7 cells to digest the external proteins, allowing the cells to lift off from the culture dish. This process is necessary for passaging cells to new plates for further growth and experimentation.

💡Cell Confluence

Cell confluence refers to the density of cells in a culture, measured by the percentage of the culture surface covered by cells. The video discusses the importance of monitoring confluence, as cells should not be allowed to reach 100% confluence to prevent contact inhibition, a phenomenon where cells stop dividing when they come into contact with each other. Proper confluence management is crucial for maintaining healthy cell growth.

💡Passaging

Passaging is the process of transferring cells from one culture vessel to another to allow for continued growth and expansion of the cell population. The video describes the steps involved in passaging, including removing the media, rinsing with PBS, treating with trypsin-EDTA, and then diluting the cells in fresh media before transferring them to new plates.

💡Contact Inhibition

Contact inhibition is a phenomenon where cells stop proliferating when they come into contact with other cells, due to the upregulation of certain pathways that signal the cells to stop dividing. The video mentions that to prevent contact inhibition, cells are passaged before they reach 100% confluence, ensuring that they have space to grow and divide without impediment.

Highlights

Introduction to cell culture basics by a molecular researcher at the University of Toronto.

Use of MCF7 breast cancer cell line for in vitro cell culture demonstration.

Emphasis on maintaining a sterile working environment in cell cultures.

Explanation of how air flow hoods provide a barrier against external contaminants.

Utilization of ultraviolet lights to irradiate the working surface and prevent contamination.

Importance of cell culture media for providing essential nutrients and balance for cell survival.

Supplementation of media with 10% fetal bovine serum for growth factor enrichment.

Use of penicillin and streptomycin to prevent bacterial contamination in cell cultures.

Preparation of cell culture bottles using a 75% ethanol solution for disinfection.

Introduction of phosphate-buffered saline (PBS) for washing cell remnants.

Use of trypsin-EDTA as a digestive enzyme to detach cells from culture plates.

Preparation of reagents in a cell culture hood to ensure sterility.

Description of cell culture vessels, including dishes and flasks, and their respective uses.

Function of a cell culture incubator in maintaining optimal growth conditions.

Process of passaging MCF-7 cells from the incubator to the flow culture hood.

Technique of removing media, rinsing with PBS, and applying trypsin to passage cells.

Dilution of cells in new media and replating for cell growth in different sized plates.

Concept of cell confluence and its measurement in cell culture.

Discussion on contact inhibition and its impact on cell health at high confluence.

Preparation for treating cells with estradiol or vehicle post-passaging.

Procedure for treating cells with calculated dilutions and allowing growth over time.

Transcripts

hello and welcome my name is Laura I am

a molecular researcher here at the

University of Toronto today we will

introduce you to the basics of cell

culture cell culture is the growth of

eukaryotic cells in vitro for scientific

purposes today we are using the mcf7

breast cancer cell line to grow up

culture harvest and treat so that you

can see them for your lab purposes one

of the most important things about

working in cell cultures that we

maintain a sterile working environment

in order to achieve this we use air flow

hoods like this air flow hoods provide a

barrier against the external environment

because if things like mold or bacteria

were to get into your samples they would

invalidate the results and you would

need to start again airflow hoods work

by generating a barrier between the

external environment and the internal

working environment how they do this is

there is a current of air that is pulled

down into the flow hood and then back

through a system of filters so that the

air in the hood itself is sterile what

we will be doing is we also use

ultraviolet lights that are mounted on

the back of the hood and those lights

irradiate the working surface so that

there is no potential for contamination

the first and most essential reagent

that we will be using is cell culture

media in this case we're using DM a.m.

or del becos modified eagle medium this

media provides salt balance pH balance

and essential nutrients so that the

cells can survive we also supplement

this media with 10% fetal bovine serum

fetal bovine serum is the plasma from a

fetal cow and what we do is we harvest

that because it contains a large number

of growth factors that will then

stimulate our breast cancer cell lines

to grow we also use a 1% prophylactic

administration of penicillin and

streptomycin what that does is it

prevents any potential bacterial

contamination that may occur in order to

prep this bottle we spray it using a 75%

ethanol solution

that disinfects the surface of the

bottle and it is now ready to be placed

in the cell culture hood the next

reagent we are using is one x phosphate

buffered saline this is just a basic

saline that is buffered to pH 7.3 that

we can use to wash off any lingering

remnants of media that may be on the

cells again it needs to be disinfected

finally the last reagent is one times

trypsin EDTA this is a digestive enzyme

that digests the external cellular

proteins so that the cells lift up from

the bottom of the plate and are in

suspension from that we can then dilute

them and replate them in different

plates that is called passages else

now that our reagents are warmed and

disinfected and we've placed them in the

cell culture hood we are ready to begin

the process of cell culture the mcf-7

breast cancer cell lines are adherent

cells meaning that they adhere to the

bottom of the culture vessel that they

are being grown in as you can see there

are multiple examples of culture vessels

this one here is a cell culture dish the

lid comes off and there are multiple

sizes that can be used depending on the

nature of your experiment this is a cell

culture flask cell culture flasks as you

can see are fully sealed and they have a

lid there are pros and cons to both the

dish and the flask dishes are easier to

work with but there's a higher potential

for contamination as the lid comes right

off flasks are a little more awkward to

work with but they are protective of the

cells because they have a lid for the

purposes of this video we will be using

the cell culture dish this is a cell

culture incubator it maintains

conditions of 37 degrees Celsius and 5%

co2 which are similar to the internal

conditions of our bodies our cells are

growing in a cell culture dish with

inside let's take a look

as you can see there are many cell types

being cultured in this incubator our

cells are the mcf7 cells and now that we

have them from the incubator we can take

them and passage them in the flow

culture hood now we are ready to passage

our mcf-7 cells for our experiment how

we do this is we will remove the

existing media rinse the cells with PBS

and then place trypsin on the cells the

trypsin will digest the external

proteins of the cells allowing them to

lift up off the bottom of the dish then

we will dilute the cells in new media

and place the newly diluted cells into

new plates that contain new media so

that the cells can now grow in different

sized plates for our experiment

initially after the cells have been

passaged

they will be quite separated and will

have a lot of space around them the

population density of cells in your

plate is called cell confluence

confluence is measured subjectively by

comparing the amount of space

surrounding the cells if the cells cover

30% of the plate then the plate is 30%

confluent if there is virtually no space

around the cells then the plate is 100%

confluent it is not a good idea to let

cells reach 100% confluence generally

when cells are too close together a

phenomenon called contact inhibition

comes into play a poptart pathways are

then up regulated in the cells in the

cells and they begin to die in order to

prevent this we passage the cells before

they reach a hundred percent confluence

now that we've passaged ourselves we put

them back into the incubator and allow

them to adhere back to the bottom of the

plate once the cells have adhered then

we can treat them with our estradiol and

or vehicle and that's what we will be

doing next so in order to treat them we

have previously calculated dilutions of

ethanol or it's vehicle aliquot it in

media these alec watts then get put into

the plates and allowed to grow for the

appropriate time limits 12 24 and 48

hours