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Cell Culture 101 1
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Undergraduate biology students and new laboratory researchers needing a practical introduction to sterile cell culture techniques and maintenance.
TL;DR
This tutorial covers the fundamentals of eukaryotic cell culture using MCF7 breast cancer cells. It explains essential laboratory practices, including maintaining a sterile environment, preparing reagents like media and trypsin, and the process of passaging cells to ensure healthy growth and experimental accuracy.
Key Takeaways
- Maintaining a sterile environment is critical in cell culture, as contamination from mold or bacteria invalidates experimental results and necessitates restarting the entire process from the beginning.
- Airflow hoods utilize filtered air and ultraviolet light to create a sterile barrier, protecting cell samples from external environmental contaminants during the handling and experimental process.
- Cell culture media, such as DMEM, provides essential nutrients, salt, and pH balance, while supplements like fetal bovine serum supply growth factors necessary for stimulating cell proliferation.
- Trypsin-EDTA is a digestive enzyme used to detach adherent cells from culture vessels by breaking down external cellular proteins, allowing researchers to passage and redistribute cells into new plates.
- Cell culture incubators mimic internal body conditions by maintaining a constant temperature of 37°C and a 5% CO2 concentration, which are optimal for the survival and growth of eukaryotic cells.
- Cell confluence refers to the percentage of a plate's surface covered by cells; researchers must monitor this to avoid overcrowding, which triggers contact inhibition and leads to programmed cell death.
- Passaging involves diluting and transferring cells to new vessels before they reach 100% confluence, ensuring they have sufficient space to grow and preventing the negative effects of high population density.
In This Video
- 00:00Introduction to Cell Culture Basics
Laura introduces cell culture as the in vitro growth of eukaryotic cells, specifically using the MCF7 breast cancer cell line.
- 00:29Maintaining a Sterile Working Environment
Airflow hoods and ultraviolet lights are essential tools used to prevent contamination from mold or bacteria during cell culture procedures.
- 01:19Essential Reagents for Cell Growth
Key reagents like DMEM media, fetal bovine serum, antibiotics, PBS, and trypsin are prepared and disinfected for use in the hood.
- 03:17Culture Vessels and Incubator Conditions
Cells are grown in dishes or flasks within an incubator that mimics internal body conditions of 37°C and 5% CO2.
- 04:41The Cell Passaging Process
Passaging involves removing old media, washing with PBS, and using trypsin to detach cells for transfer into new culture vessels.
- 08:21Monitoring Confluence and Cell Health
Cell confluence is monitored to prevent contact inhibition and apoptosis, ensuring cells are passaged before they become too crowded.
- 09:13Treating Cells for Experimental Analysis
After passaging and adherence, cells are treated with specific compounds like estradiol to study their growth over 12 to 48 hours.
Questions & Answers
What is cell culture?
Cell culture is the process of growing eukaryotic cells in vitro for scientific research purposes.
Why are airflow hoods used in cell culture?
Airflow hoods provide a sterile working environment by using filtered air and ultraviolet lights to prevent contamination from mold or bacteria, which would invalidate experimental results.
What is the purpose of fetal bovine serum in cell media?
Fetal bovine serum is added to cell culture media because it contains growth factors that stimulate the growth of cell lines, such as MCF7 breast cancer cells.
What does trypsin do to cells?
Trypsin is a digestive enzyme that breaks down external cellular proteins, allowing adherent cells to detach from the culture vessel and enter suspension for passaging.
What is cell confluence?
Cell confluence refers to the population density of cells in a plate, measured by the percentage of the surface area covered by the cells.
Why should you avoid 100% cell confluence?
At 100% confluence, cells experience contact inhibition, which triggers apoptotic pathways and causes the cells to die.
Key Terms
- Passaging — The process of diluting and transferring cells from an existing culture vessel into new plates to allow for continued growth.
- Adherent cells — Cells that require a surface, such as the bottom of a culture dish or flask, to attach to in order to grow.
- Contact inhibition — A phenomenon where cells stop growing and begin to die when they become too crowded and touch one another.
- DMEM — Dulbecco's Modified Eagle Medium, a standard reagent providing essential nutrients, salt, and pH balance for cell survival.