Cell Culture 101 1
Summary
TLDRIn 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
🔬 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.
🌱 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.
Mindmap
Keywords
💡Cell Culture
💡Sterile Environment
💡Airflow Hood
💡Cell Culture Media
💡Fetal Bovine Serum
💡Penicillin and Streptomycin
💡Phosphate-Buffered Saline (PBS)
💡Trypsin-EDTA
💡Cell Confluence
💡Passaging
💡Contact Inhibition
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
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