Lecture 3 Animal Culture PDF

Culturing and Subculturing of Animal Cells Dr Mohd Nazmi Bin Abd Manap  Subculture refers to the process of transferring a portion of an existing cell culture into a new culture vessel to facilitate cell growth and expansion. De...

Culturing and Subculturing of Animal Cells Dr Mohd Nazmi Bin Abd Manap  Subculture refers to the process of transferring a portion of an existing cell culture into a new culture vessel to facilitate cell growth and expansion. Definition of Involves the removal of cells from the Subculture original culture vessel and their transfer into fresh growth medium or onto a new substrate to create a new subculture. Subculture is usually done when cells have occupied most of the space available. Importance of subculture Essential for maintaining Prevent overcrowding/overgrowth of cells healthy and proliferating Allows for the removal of accumulated waste products dead cells cell populations over Provide fresh nutrients and growth factors to support continued time by: cell growth Enable propagation cells To generate larger cell population with enough cell numbers for for experimental downstream assays/application purpose Factors influencing subculturing frequency Growth characteristics of specific cell types The density of cells required/confluency For suspension usually involves dilution of cell culture to main optimal density. Routine maintenance of cell line Media Changes: Essential to provide fresh nutrients and growth factors, remove waste products, and maintain optimal pH and osmolality conditions. The frequency depends on the cell type and growth rate, and it is important to avoid prolonged exposure to low-nutrient or acidic conditions. Some cells require you to change media every 3 days (slowly proliferating cells) and subculture every 7 days because the nutrient in the media have been exhausted. Subculturing: As discussed earlier, is the process of transferring a portion of the cell culture into a new culture vessel to promote cell growth and prevent overcrowding. It involves detaching adherent cells or diluting suspension cells into fresh growth medium. Performed when the cells reach a desired confluency or cell density. Routine maintenance of cell line Cell Counting: Allows for the assessment of cell density and viability. It can be performed using various methods such as manual counting using a hemocytometer or automated cell counters. Helps determine the appropriate seeding density for subculturing and enables monitoring of cell growth rates and population doubling times. Sterility and Contamination Control: Maintaining aseptic conditions and preventing contamination is crucial for cell line maintenance. Involves working in a laminar flow hood, using sterile techniques, and regularly testing for potential contaminants such as bacteria, fungi, and mycoplasma. Sterility checks can be performed using culture media or specific tests such as polymerase chain reaction (PCR) Routine maintenance of cell line Cryopreservation and Thawing: Cryopreservation allows for long-term storage of cell lines by freezing them in liquid nitrogen or ultra-low temperature freezers. Regularly cryopreserving cell stocks helps maintain the stability and genetic integrity of the cell line. Thawing of cryopreserved cells should be done carefully to ensure high post-thaw viability and recovery. Record-Keeping: Maintain accurate and detailed records of cell line maintenance activities, including passage numbers, media formulations, subculturing dates, cell counts, and any observed changes or issues. A good record-keeping measures facilitates traceability, troubleshooting, and maintaining consistency in experimental protocols. Routine maintenance of cell line Authentication and Quality Control: Important to ensure their identity and prevent cross-contamination. Methods include short tandem repeat (STR) profiling, isoenzyme analysis, and DNA sequencing. Quality control measures such as mycoplasma testing, and periodic monitoring of cell line behavior also help maintain the reliability and reproducibility of experimental results. Replacement Required when: pH drop: most cell will stop growing of medium when pH falls from 7.0 to 6.5 and lose viability if it drop further to 6.0. Cell at high concentration: more cells more often you need to change medium as nutrient is exhausted from the use. Cell type: some cells are much faster than others in using the medium Morphological deterioration: if observed must change to stop it from being irreversible as cells will enter apoptosis Normal growth rate of cells after subculture After seeding, there will be a lag period followed by a log phase basically when cells will grow exponentially. Feeding (medium change) usually performed during exponential growth. Cell concentration at subculture General rule of thumb, most continuous cell lines subculture satisfactorily at a seeding concentration between 1 × 104 and 5 × 104 cells/mL Finite fibroblast cell lines subculture at about the same concentration, and more fragile cultures, such as endothelium and some early-passage epithelia, subculture at around 1 × 105 cells/mL.  Usual ratio of medium volume to surface area 0.2-0.5ml/cm2 Volume Optimum ratio depends on the oxygen requirement of the cells Depth and Cells with high oxygen requirement do better in shallow medium (2mm) Surface Area Cells (5mm) with low oxygen requirement better in deep medium Depth with more than 5mm will affect gaseous diffusion and may become a limiting factor. Medium volume for subculturing  1. Prewarm materials at 37° on waterbath General Protocol for Including trypsin, medium PBS(phosphate buffer saline) subculturing 2. Examine cells inside flask using inverted microscope make sure it is healthy monolayer of cells 3. Remove old medium from flask 4. Rinse with PBS 5. Remove PBS 6. Add trypsin solution 7. Incubate at 37° for 2-3 mins- examine cells for detachment 8. Add complete growth media (2 volume) to inactivate trypsin 9. Transfer cell suspension to a falcon tube and centrifuge at 300- 1000 X g for 5-10 min. 10. Remove supernatant and add complete growth medium and resuspend 11. Count cell density using hemocytometer or automated cell counter 12. Dilute remaining solution for seeding purpose (subculture in new flask). This Photo by Unknown Author is licensed under CC BY-SA  https://www.sigmaaldrich.com/MY/en /technical-documents/protocol/cell- culture-and-cell-culture- analysis/mammalian-cell-culture/cell- dissociation-with-trypsin How to subculture (passage) primary cells? https://youtu.be/k2PMEET4Smo Contamination in Cell Culture Dr Mohd Nazmi Bin Abd Manap Source of contamination 1. Operator Techniques: Improper Aseptic Techniques: Issue: Failure to maintain proper sterile techniques during cell handling and manipulation. Prevention: Training operators in aseptic techniques, wearing appropriate personal protective equipment (PPE), and regular reinforcement of good laboratory practices. Source of contamination 2. Environment: Airborne Contaminants: Issue: Airborne particles carrying bacteria, fungi, or viruses settling onto open cultures. Prevention: Use of laminar flow hoods, regular air quality monitoring, and maintaining a clean laboratory environment. Cross-contamination: Issue: Movement of personnel between different work areas without proper decontamination. Prevention: Implementing proper protocols for equipment and personnel movement, and designating specific areas for different tasks. Source of contamination 3. Use of Laminar Flow: Maintenance Issues: Issue: Inadequate maintenance leading to compromised airflow and filtration. Prevention: Regular maintenance and certification of laminar flow hoods, and prompt repair of any issues. Limited Workspace: Issue: Overcrowded workspaces inside laminar flow hoods may lead to contamination. Prevention: Strict adherence to recommended workspace capacity, and organizing work areas efficiently. Source of contamination 4. Cold Stores: Temperature Fluctuations: Issue: Fluctuations in temperature inside cold storage affecting the integrity of stored materials. Prevention: Regular monitoring and maintenance of cold storage units, and proper organization of materials to allow for proper airflow. Cross-contamination in Storage: Issue: Improper storage leading to cross-contamination between different cell lines or materials. Prevention: Strict labeling, segregation, and organization of stored materials, as well as regular cleaning and maintenance. Source of Contamination 5. Sterile Materials: Quality of Sterile Materials: Issue: Use of contaminated or improperly sterilized materials. Prevention: Quality control of purchased sterile materials, proper autoclaving procedures, and adherence to expiration dates. Handling Procedures: Issue: Mishandling of sterile materials during storage or use. Prevention: Proper training of personnel in the handling and storage of sterile materials, and use of aseptic techniques. Source of contamination 6. Cell Line: Contaminated Cell Lines: Issue: Introduction of cell lines contaminated with bacteria, fungi, mycoplasma, or viruses. Prevention: Regular authentication of cell lines, quarantine of newly acquired cell lines, and strict adherence to recommended culture conditions. Cross-contamination between Cell Lines: Issue: Cross-contamination between different cell lines leading to misidentification. Prevention: Regular monitoring, proper labeling, and adherence to cell line authentication protocols. Types of contamination in Animal Cell Culture Bacteria: Fungi: Mycoplasma: Virus: Common contaminants Yeasts and molds can Mycoplasma Viral contamination can include Gram-positive infiltrate cultures. contamination is a occur, posing a threat to (e.g., Staphylococcus, Detected through significant concern as cell cultures. Bacillus) and Gram- microscopic examination, these small bacteria lack a Detection involves negative bacteria (e.g., visual inspection, and cell wall. molecular methods like Escherichia coli). culture on specialized Detected using PCR, PCR, virus-specific assays, Detected through fungal media. ELISA, or staining or cytopathic effect microscopic examination, methods. observation. Gram staining, and culture on selective media. Detection of Microbial Contamination in Animal Cell Culture: 1. Microscopic Examination: 1. Cells are observed under a microscope for signs of contamination, such as irregular morphology or unusual structures. 2. Cultural Methods: 1. Cells are cultured on specific media to allow the growth of contaminants for identification. 3. Nucleic Acid-Based Methods: 1. PCR (Polymerase Chain Reaction) is widely used to detect microbial DNA and RNA. 4. Immunological Methods: 1. ELISA (Enzyme-Linked Immunosorbent Assay) can be employed for the detection of specific microbial antigens. Types of contamination in Animal Cell Culture Bacterial Contamination Bacteria are much smaller than eukaryotic cells. Appear as dark rod-like structures, spheres or spiral structures under the microscope, exist as single cells, in pairs, chains, or clusters. Can be visualized using phase contrast and 100x - 400x magnification. Phase contrast facilitates detection, especially at low contamination levels.  Types of contamination in Animal Cell Culture Mycoplasma contamination 0.1 - 0.3 µm in diameter, therefore detection via brightfield microscopy is not possible. This lack of visible signs of infection increases the risk of mycoplasma-positive cells remaining unnoticed. bacteria that lack a cell wall, and they are considered the smallest self-replicating organism. Because of their extremely small size (typically less than one micrometer), mycoplasma are very difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; Detection is possible using fluorescence kit  Types of contamination in Animal Cell Culture Yeast Contamination Can be visualized using phase contrast at 100x - 400x magnification. Phase contrast facilitates detection, especially at low contamination levels. Yeasts appear as ovoid bright particles between the cells. They can exist as single cells or in in the form of chains or branches. Oberservable Signs of Contamination 1. Changes in Cell Morphology: Contaminants can alter the morphology of the cultured cells. This may include changes in cell shape, size, and overall appearance under a microscope. 2. Presence of Floating Debris: Contaminated cultures may show the presence of floating particles or debris, which can be visible upon microscopic examination. 3. Cell Clumping: Some contaminants can cause cells to clump together, leading to altered cell distribution and adherence patterns. 4. Unusual Color Changes: Changes in the color of the culture medium or the cells themselves may indicate contamination. For example, media can turn yellow due to bacterial contamination. (pH changed) 5. Cloudiness in Culture Medium: A previously clear culture medium becoming cloudy can be a sign of microbial contamination. This is particularly true for bacterial contamination. 6. Slow or Altered Growth: Contaminants can affect cell proliferation, leading to slower or abnormal growth patterns compared to healthy cultures. Many contaminants, such as mycoplasma and certain viruses, are not visible through routine microscopic examination. Therefore, regular testing using specific assays, such as PCR or ELISA, is essential for detecting these types of contaminants. Cross contamination in cell culture Refers to the inadvertent transfer of biological material, such as cells or microorganisms, from one culture to another. This can occur between different cell lines, experimental samples, or cultures in a laboratory setting. Cross contamination can compromise the integrity of experiments, research, and the reliability of results. Cross contamination in cell culture Cell with different morphology grows together as a result of failure failure to adopt good cell culture practices Cross contamination in cell culture Key aspects of cross contamination in cell culture include: 1.Transfer of Cells: Unintentional transfer of cells from one culture vessel to another. This can occur through contact with contaminated surfaces, equipment, or even through the air. 2.Misidentification of Cell Lines: Cross contamination may lead to misidentification of cell lines. This is particularly problematic when different cell lines have distinct characteristics or when the identity of a specific cell line is critical to the experimental design. Cell Culture Contamination and Quality Control https://youtu.be/tuJ8VWvX01M?si=R3i8hr6ekg0nk5Go