Adherent Cell Culturing Using Mammalian Cell Lines | PraxiLabs

Adherent Cell Culturing using Mammalian Cell Lines

Biology | Toxicology | Biochemistry | Proteomics | Pharmacology

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General Aim

This experiment aims at showing how cells are cultured, propagated, sub-cultured, and froze in liquid nitrogen for long-term storage. In Vitro Cell Culturing is a very important protocol for preparing cells needed for all subsequent In Vitro Cytotoxicity and Genotoxicity assays.


In Vitro Cell Culturing using Adherent Mammalian Cell Lines

Learning Objectives (ILO’s)

  • Successfully handle the required instruments and consumables needed in the cell culturing and sub-culturing.

  • Work and follow the general safety guidelines for Good Laboratory Practice (GLP).

  • Strictly work and follow the Aseptic Techniques of cell culturing.

  • Thaw cells from Liquid Nitrogen and seed them in cell culture flasks.

  • Check the confluence, harvest cells, and count them microscopically.

  • Scaling-up the cultured cells for the setting of further experiments.

  • Freezing cells in Liquid Nitrogen for long-term storage

Theoretical Background/Context

Cell culture is the growth of cells from an animal in an artificial, controlled environment. Cells are removed either from the organism directly and disaggregated before cultivation or from a cell line or cell strain that has previously been established.

Certain culture conditions depend on the cell type; however, each culture must consist of a suitable vessel with a substrate or medium that supplies the nutrients (such as amino acids, carbohydrates, vitamins, and minerals), growth factors, or essential hormones for culturing cells. Gases (O2, CO2), and physicochemical environment (pH, osmotic pressure, temperature) also play an important role to regulate the proper cell growth in an artificial environment

Principle of Work

Cell culture is an amazing tool that allows for easy control and manipulation of all physiochemical and physiological cell factors, such as temperature, osmotic pressure, pH, gas, hormones, media, and its supplements. Media supplements help to optimize cell growth for specific applications depending on the chosen tissue or cell type. To be successful in cell culture, it is essential to remain in a contamination-free environment (bacteria, fungi, mycoplasma …. etc.), so following aseptic techniques ensure that no microorganisms enter the cell culture.

In this protocol, adherent cell lines will be first thawed from liquid nitrogen and seeded in a cell culture flask. Following 24 hours, cells will be visualized by light microscope for confluency, in order to define the best time for sub-culturing and scaling-up of cells. Once the cells exceed 90% confluency, they will be sub-cultured and passaged into other culture vessels. For long-term storage, cells will be frozen in a special freezing medium before storing them in liquid nitrogen.

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