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Cytion's Guide to Cell Culture Excellence

Cytion provides comprehensive guidelines for the culturing of cell lines, emphasizing the importance of sterility and aseptic techniques. Our protocols ensure successful cell culture endeavors across a range of cell types and applications.

1. Laboratory Design

Designing a tissue culture laboratory requires thoughtful planning to ensure the production of high-quality material in a safe and efficient environment. Optimal facilities include distinct areas for quarantine and processing of uncontaminated materials, with dedicated incubators for each. When space does not permit separation by area, temporal separation of handling different materials is advised. Strict cleaning protocols and adherence to at least Category 2 containment levels are essential to minimize contamination risks and maintain a controlled laboratory environment.

2. Establishing an Aseptic Workspace

  • Hood Practices: Maintain laminar airflow by correctly positioning the hood sash and avoiding clutter.
  • Sterilization: Autoclave tools like pipette tips and glass pipettes, and use 70% ethanol for surface decontamination.

3. Media and Reagent Preparation

  • Culture Media Selection: Consult with our detailed media charts to match your cell line with the appropriate DMEM or RPMI media, supplemented with additives such as Fetal Bovine Serum (FBS) and glutamine.
  • Sterility of Supplements: All culture media and supplements must be sterile, employing filter sterilization if necessary.
  • Personal Protective Equipment: In cell culture labs, minimizing harm relies on strictly adhering to personal protective gear, such as gloves compliant with the EN374-3 standard.
  • Desinfection: Minimizing harm is also dependent upon the correct use of disinfectants such as Sodium Hypochlorite or Ethanol. For comprehensive safety and effectiveness in laboratory hygiene, the following table details additional disinfectants and their specific use cases:


Effective Against




Sodium Hypochlorite

Broad spectrum, including viruses

1000ppm for surfaces, 2500ppm for pipettes, 10,000ppm for waste/spillages

Corrosive to metals, inactivated by organic matter

Should be made fresh daily, not used on metal surfaces


Bacteria, most viruses


Not effective against non-enveloped viruses

Use in well-ventilated areas, avoid prolonged skin contact




Not effective against viruses

Use in well-ventilated areas, avoid prolonged skin contact


Broad spectrum, used for fumigation

Varies (used in vaporized form for fumigation)

Irritant, sensitizing, requires ventilation

Avoid exposure, remove hypochlorites before fumigation

 4. Culturing Environment Setup

  • Flasks for Adherent Cell Lines: Tissue-culture treated flasks are generally adequate for the majority of cell lines. For certain cell types that require additional support, flasks may be pre-treated or coated with substrates such as gelatin or fibronectin. These coatings can significantly improve cell attachment and proliferation. Detailed protocols for preparation and coating can be found on the respective product information sheets.
  • Flasks for Suspension Cell Lines: Use flasks specifically designed for non-adherent cells, which allow for free movement and adequate gas exchange. These flasks do not require a surface treatment that promotes cell attachment.

5. Monitoring Cell Health

Maintaining the health of cell cultures is a critical aspect of cell culture work. Continuous monitoring is essential to ensure the integrity and reproducibility of experimental results. Here are detailed practices for monitoring cell health:

5.1. Daily Checks

  • Microscopic Evaluation: Inspect cells daily using a microscope to assess key indicators of cell health, including consistent cell attachment, characteristic morphology, and expected growth patterns. Look for uniformity in cell size and shape, the presence of distinct nuclei, and the absence of granularity that may indicate cell death.
  • Growth Rate Monitoring: Keep track of the proliferation rate to ensure it aligns with the expected doubling time for the cell line. Sudden changes in growth rate can signal an underlying issue with cell health or culture conditions.
  • Medium Assessment: Check the color of the culture medium, which can indicate pH changes. A yellowing medium suggests increased acidity, often a byproduct of cellular metabolism or bacterial contamination, while a purple or pink hue can indicate a more basic environment.

5.2. Criteria for Discarding Cultures

  • Contamination indicators: Be vigilant for signs of contamination such as turbidity in the medium, unexpected pH changes, or the presence of microbial colonies. Contaminations can be bacterial, fungal, or viral, and each type has distinct visual cues, such as bacterial films or fungal hyphae.
  • Abnormal Morphology: If cells exhibit persistent abnormal changes in morphology not associated with normal growth or differentiation, it may be necessary to discard the culture. This includes extensive cell rounding, detachment, or the presence of cellular debris.
  • Irreversible Stress Signs: Look for indications of irreversible stress or toxicity, such as vacuolation, membrane blebbing, or apoptotic bodies. These are often precursors to cell death and can affect experimental outcomes.
  • Degradation of Growth Conditions: If the culture has reached confluence or overgrowth, leading to nutrient depletion and waste accumulation, the culture should be subcultured or discarded to avoid negative effects on cell health.

6. Subculturing Strategies

Subculturing, or splitting cells, is a routine part of cell culture that involves transferring a portion of a cell culture to fresh growth medium to propagate the culture. Careful planning and execution are critical to the success of this process. Here are some enhanced strategies for effective subculturing:

Planning Splits

  • Optimal Split Ratios: Determine the ideal split ratios based on cell line characteristics, growth rates, and the intended use of the cultures. This might involve a 1:2 split for rapidly growing cells or a 1:10 split for slower-growing lines.
  • Vendor Specifications: Refer to the vendor’s product sheets for recommendations specific to each cell line, which often include detailed protocols for subculturing and the conditions that the cells require.
  • Continuity of Culture: Maintain a master cell bank and use a consistent subculturing routine to ensure the longevity and genetic stability of the cell line over time.

Splitting Adherent Cells

For a detailed guide on splitting adherent cells, please refer to our separate article below:

Cell Culture Dissociation ->

7.        Media Maintenance

Timely Nutrient Replenishment: Media should be changed before cells deplete essential nutrients, which is crucial for their growth and metabolic functions.

pH and Toxin Control: Regular changes prevent the accumulation of metabolic byproducts and maintain a physiological pH, critical for cell health.

8.       Passage Number Control

Passage Limitation: Keep a detailed log of cell passages. Frequent passaging can lead to genetic drift, which may alter cell phenotype and behavior. By limiting the number of passages, you maintain the genetic stability and integrity of the cell line.

Documentation of Passage Number: Each time cells are subcultured, record the passage number. This information is essential for tracking cell line history and identifying potential passage-related changes in cell behavior. Consult our guide below for insights on accurately tracking passage numbers.

Passage number Guide ->

9.       Ensure Cell Line Integrity

  • Verification Records: Regularly verify cell line identity (e.g., through short tandem repeat (STR) profiling) and annotate this in the records to ensure that the correct cell line is being used throughout experiments.
  • Mutation Monitoring: If possible, monitor for key genetic or phenotypic changes that may indicate genetic drift or cell line contamination, and maintain these records for reference.


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