Cell Culture Techniques Quiz

Culturing and Subculturing of Animal Cells

• Subculture is the process of transferring a portion of an existing cell culture to a new vessel to promote growth.
• It involves removing cells from the original vessel and transferring them to fresh growth medium or a new substrate.
• Subculturing is typically performed when cells have occupied most of the available space.

Importance of Subculture

• Preventing overcrowding and overgrowth of cells
• Removing accumulated waste products from dead cells
• Providing fresh nutrients and growth factors to support continued cell growth
• Enabling cell propagation for experimental purposes, creating larger populations with sufficient numbers for downstream assays or applications.

Factors Influencing Subculturing Frequency

• Growth characteristics of specific cell types
• Required cell density/confluency
• For suspension cultures, dilution of cell culture to maintain optimal density is often required.

Routine Maintenance of Cell Lines

Media Changes

• Essential to provide fresh nutrients and growth factors
• Maintain optimal pH and osmolality
• The frequency depends on the cell type and growth rate
• Prolonged exposure to low-nutrient or acidic conditions should be avoided
• Some cells require media changes every 3-7 days depending on proliferating rate.

Subculturing

• The process of transferring a portion of cell culture into a new vessel.
• The process promotes cell growth and prevents overcrowding.
• Involves detaching adherent cells or diluting suspension cells in fresh growth medium.
• Performed when cells reach desired confluency or density.

Cell Counting

• For assessing cell density and viability
• Methods include manual counting (e.g., hemocytometer) or automated cell counters.
• Helps determine appropriate seeding density for subculturing
• Monitors cell growth rates and population doubling times.

Sterility and Contamination Control

• Maintaining aseptic conditions is crucial to prevent contamination.
• Involves working in a laminar flow hood, employing sterile techniques, and routinely testing for contamination
• Examples of contamination include bacteria, fungi, and mycoplasma.
• Sterility checks may use culture media or specific tests like polymerase chain reaction (PCR).

Cryopreservation and Thawing

• Cryopreservation allows long-term storage of cell lines.
• Freezing cells in liquid nitrogen or ultra-low-temperature freezers.
• Regular cryopreservation helps maintain cell line stability and genetic integrity.
• Thawing cryopreserved cells should be done carefully for optimal post-thaw viability and recovery.

Record-Keeping

• Maintaining accurate records of cell line activities (passage numbers, media, subcultures, dates, cell counts)
• Good record keeping helps with traceability, troubleshooting.
• Ensures consistency in experimental protocols.

Authentication and Quality Control

• Ensuring cell identity and preventing cross-contamination.
• Methods include short tandem repeat (STR) profiling, isoenzyme analysis and DNA sequencing.
• Implementing quality control measures, such as, mycoplasma testing, and regularly monitoring cell line behavior.
• This aids in the reliability and reproducibility of experimental results.

Replacement of Medium

• Required when pH drops below a certain level (7.0 to 6.5), cell viability will be lost.
• High cell concentration necessitates changes for nutrient exhaustion
• Some cell types consume medium faster than others.
• Morphological deterioration or apoptosis may necessitate changing the medium.

Normal Growth Rate of Cells After Subculture

• After seeding, there is a lag phase followed by an exponential growth phase
• Feeding (medium change) is usually performed during exponential growth.

Cell Concentration at Subculture

• Most continuous cell lines are subcultured at seeding concentrations between 1 x 10⁴ to 5 x 10⁴ cells/mL
• Finite fibroblast cells are subcultured at similar concentrations
• Fragile cultures (endothelium, early passage epithelial) are subcultured at around 1 x 10⁵ cells/ml.

Volume, Depth, and Surface Area

• Usual ratio of medium volume to surface area is 0.2-0.5 mL/cm².
• Oxygen requirement of cells affects the optimum ratio
• Cells with high oxygen requirements perform better in shallow media (2mm), while celles with low oxygen requirements perform better in deep media (5mm).
• Depth exceeding 5mm can affect gaseous diffusion and might limit growth.

Medium volume for subculturing

• Appropriate medium volumes for various flask sizes are listed, correlating area size to volume e.g. 25cm² flask = 5-10 mL; 75 cm² flask = 10-30mL

General Protocol for Subculturing Monolayer of Cells

• Detailed steps, from pre-warmed materials to the final dilution of the remaining solution to seed new flasks.

How to Subculture (Passage) Primary Cells?

• Detailed Video Protocol (link provided)

Contamination in Cell Culture

Source of Contamination (General)

• Operator techniques that include improper aseptic techniques.
• Environmental factors such as airborne contaminants (bacteria, fungi, or viruses).
• Cross-contamination, through movement of personnel between different work areas without proper decontamination.
• Issues with maintaining laminar flow hoods.
• Cold storage fluctuations.
• Quality of sterile materials, contaminated or improperly sterilized materials.
• Contaminated cell lines, that can include bacteria, fungi, mycoplasma, or viruses.

Contamination Source Details (Specific)

• Operator Techniques: Issues include failure to maintain proper sterile techniques during handling and manipulation. Prevention involves training in aseptic techniques, appropriate personal protective equipment (PPE), and reinforcement of good laboratory practices.
• Environment: Issues include airborne particles carrying bacteria, fungi, or viruses settling onto open cultures Prevention includes laminar flow hoods, regular air quality monitoring, and maintaining a clean laboratory environment.
• Cross-Contamination: Issues from personnel movement between work areas without proper decontamination. Prevention involves implementing protocols for equipment and personnel movement, and designating specific areas for different tasks.
• Use of Laminar Flow: Issues include inadequate maintenance leading to compromised airflow and filtration. Prevention involves regular maintenance and certification of laminar flow hoods, any needed repairs, adhering to recommended workspace capacity, and organizing work areas efficiently.
• Cold Stores: Issues include temperature fluctuations affecting stored material integrity and requiring careful material organization to maintain proper airflow. Prevention includes regular monitoring, maintaining cold storage units, and appropriate material storage to prevent cross-contamination, strict labeling, segregation, storage and regular cleaning and maintenance.
• Sterile Materials: Issues include use of contaminated or improperly sterilized materials, necessitating quality control of purchased sterile materials, proper autoclaving procedures and adherence to expiration dates, and handling procedures, preventing mishandling of sterile materials during use by training personnel in the correct handling, storage and use of aseptic techniques.
• Cell Lines: Issues include introducing contaminated cell lines containing bacteria, fungi, mycoplasma, or viruses. Prevention includes regular authentication of cell lines, quarantine of newly acquired cell lines, adherence to recommended culture conditions, cross-contamination between cell lines leads to misidentification, preventing the issues by regular monitoring, proper labeling, and adherence to cell line authentication protocols.

Types of Contamination

• Details provided for bacteria, fungi, mycoplasma, and viruses, including detection methods.

Detection of Microbial Contamination in Animal Cell Culture

• Detailed methods for microscopic examination, cultural methods, nucleic acid-based (PCR), and immunological methods (ELISA) for detecting contamination.
• Types of contamination in bacterial contamination, mycoplasma, yeast and overall contamination observation.

Cross-Contamination in Cell Culture

• Inadvertent transfer of biological material, such as cells or microorganisms, from one culture to another.
• Reasons for cross-contamination are through contact with contaminated surfaces, equipment, or through air.
• Cross contamination can cause misidentification of cell lines, which is crucial for accurate experimental design.

Test your knowledge on cell culture practices, including subculturing, seeding concentrations, and contamination control. This quiz covers essential techniques and protocols for maintaining continuous cell lines. Challenge your understanding of the best practices in laboratory settings.