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Cell Culture: Step-by-Step Guide
April 24, 2024

Cell Culture: Step-by-Step Guide

Table of Contents

Cell Culture: Step-by-Step Guide

Cell culture is defined as a process where the cell particles of a human, animal, or insect are examined for better treatment or different purposes. The cells may be derived from multicellular eukaryotes that are already established cell lines or cell strains. The guide provides a step-by-step guide to the cell culture process. 

Application of cell culture 

  • Cell culture is one of molecular and cellular biology’s most consistent and reproducible tools. 
  • Cell culture is a model system used to diagnose certain diseases and drug testing
  • Cell culture assists in examining normal cell homeostasis, cell biochemistry, and compound effect on mutagenesis.

Step 1: Prepare your Work Station 

Before starting your cell culture process, it’s essential that you keep your workstation clean and disinfected from germs or residues. Any contamination can ruin the results of the cell culture, so you should spend time setting up equipment and surfaces thoroughly. I also recommend keeping a notebook and pencil at the side after writing the objective for the cell culture process. 

Step 2: Source your cells 

The next step is to obtain the cells for the process to be started. This could be a tissue sample, a cell bank, or a cell line. It is crucial to make sure that the cells are healthy and viable before proceeding to the next step. The health of the cell during the cell culture process needs to be checked. 

Step 3: Cell Observation 

After you have checked the cell quality and health, the next step is to perform the following observations using an optical microscope or other suitable observational device. 

  • Check for foreign objects other than cells in the vessel. 
  • You need to determine if the cells are subconfluent or over-proliferated 
  • Check if the culture is proceeding normally by analysing the cell morphology and condition. 

After the above assessments are cleared, adding fresh medium to resuspend the cell collected by centrifugation is crucial. You need to be careful when using a small amount of the medium, as the density of the cell suspension needs to be higher than the passage. The cells are suspended using a pipette while the number of cells is measured using microscope.

Step 4: Dispensing 

Once the required number of cells has been reached, pipette the same volume of the 2x concentrated cryopreservation solution to the cell suspension. Pour a fixed volume of solution into the cryotube, pipetting or swirling continuously.

Step 5: The freezing process 

To maintain a freezing rate of -1°C per minute, the cryotube needs to be put in a freezer container immediately and stored in a deep freezer (-80 °C). An alternative is to use a programmable controlled-rate freezer. Following cell freezing, the frozen cryotubes are to be kept in the vapour phase in a cryogenic deep freezer (-150 °C) or liquid nitrogen storage tank. However, the process differs according to the kind of cryopreservation solution used.

Step 6: Thawing

After thawing, culture one or two of the frozen vials. Verify if the cells can increase in the same way and display nearly the same properties as before. Stop the cell culture as soon as this is verified.

Step 7: Start of experimenting 

Prepared cell stocks should be defrosted as needed for research endeavours. Following a specific duration in culture, the cells ought to be disposed of and replaced with freshly thawed stock.


Cell culture is a complicated technique that one can master after practice. This is an important practice in molecular and cellular biology that offers a reliable platform for different applications like disease diagnosis, research, and drug testing. The step-by-step guide helps illuminate the importance of the cell culture process. 

Every step—from preparation to freezing and thawing—requires accuracy and close attention to detail to maintain the integrity of the cell culture. By adhering to these criteria, researchers can use cell culture to improve our understanding of cellular processes, disease mechanisms, and therapeutic approaches. Cell culture continues to be a fundamental component of scientific research, fostering creativity and advancement across various disciplines, provided that it is refined and best practices are followed.


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