PREPARATION OF ANIMAL CELL CULTURE MEDIA

 

EXPERIMENT NO:

TITLE: PREPARATION OF TISSUE CULTURE MEDIA

 

Introduction:

Animal tissue culture media are specially formulated solutions used to grow and maintain animal cells or tissues in vitro (outside the organism in a controlled environment). These media provide essential nutrients, growth factors, and environmental conditions needed for cell survival, growth, and function. Here are some key components and commonly used types of animal tissue culture media:

 

Basic Components of Tissue Culture Media:

• Amino Acids: Essential for protein synthesis.
• Vitamins: Act as coenzymes and antioxidants.
• Inorganic Salts: Maintain osmotic balance and membrane potential.
• Carbohydrates (e.g., glucose): Primary energy source for cells.
• Fatty Acids and Lipids: Used for membrane formation and energy.
• Serum: Provides growth factors, hormones, and other proteins.
• Buffering Agents (e.g., bicarbonate, HEPES): Maintain pH stability.
• Antibiotics: Prevent bacterial and fungal contamination.

2. Commonly Used Animal Tissue Culture Media:

• DMEM (Dulbecco's Modified Eagle Medium):
• High glucose content, suitable for a wide range of cell types.
• Contains amino acids, vitamins, salts, and glucose.
• RPMI-1640:
• Commonly used for lymphoid cells and hybridomas.
• Contains glucose, vitamins, and amino acids.
• MEM (Minimum Essential Medium):
• Widely used for a variety of mammalian cell types.
• Available in different formulations (with or without serum, with different glucose concentrations).
• F-12 (Ham's F-12):
• Developed for the growth of rodent cells and hybridomas.
• Contains a more complex mix of nutrients than MEM.

3. Specialized Media:

• Serum-Free Media:
• Formulated without serum, often supplemented with specific growth factors.
• Used for reducing variability and for cells that require defined conditions.
• Stem Cell Media:
• Tailored for the culture of stem cells.
• Contains specific factors to maintain pluripotency or direct differentiation.

 

4. Supplementary Additives:

• Fetal Bovine Serum (FBS):
• Most commonly used serum supplement, providing a wide range of growth factors.
• L-Glutamine:
• An essential amino acid often added separately due to its instability in solution.
• Insulin-Transferrin-Selenium (ITS):
• Used in serum-free cultures to support cell growth and metabolism.

These media are selected based on the specific requirements of the cells or tissues being cultured, such as the species of origin, type of cell (e.g., epithelial, fibroblast, neuronal), and the experimental goals.

Objective:

To prepare tissue culture media suitable for the growth and maintenance of animal cells, ensuring it is sterile and properly supplemented with necessary nutrients and growth factors.

Materials Required:

• Animal Cell culture media (e.g., DMEM, RPMI-1640, MEM)
• Deionized or distilled water
• Fetal Bovine Serum (FBS) or other serum
• Antibiotics (e.g., Penicillin-Streptomycin) and antifungal agents (optional)
• HEPES buffer (optional)
• pH meter or pH paper
• Sterile 0.22 µm filter units
• Sterile storage bottles
• Magnetic stir plate and stir bar
• Personal Protective Equipment (PPE): Lab coat, gloves, safety goggles

Procedure:

1.To the media add Fetal Bovine Serum (FBS), typically at a final concentration of 5-20%, depending on the cell type and experimental requirements.

2.Antibiotics/Antifungals (Optional): Add antibiotics like Penicillin-Streptomycin and antifungal agents like Amphotericin B if needed, usually at a final concentration of 1% from a 100x stock solution.

3.HEPES Buffer (Optional): If additional pH buffering is needed, add HEPES to a final concentration of 10-25 mM.

4. Sterilization: Filter the prepared media through a 0.22 µm sterile filter to remove any particulates and to ensure sterility. Use a vacuum pump or sterile syringe to facilitate filtration. Collect the filtered media in sterile bottles or containers.

5.  Labeling: Label the bottles with the media type, date of preparation, and any supplements added.

 6. Storage:

§  Refrigeration: Store the prepared media at 4°C if it will be used within a few weeks. Protect serum-containing media from light to prevent degradation.

§  Freezing (Optional): For long-term storage, aliquot the media into smaller volumes and freeze at -20°C. Avoid repeated freeze-thaw cycles.

7. Quality Control:

§ Sterility Test: Test the sterility of the media by incubating a small aliquot at 37°C for a few days and observing for any microbial growth.

§ pH Check Before Use: Recheck the pH of the media before use, especially if it has been stored for some time.

Observations:

• The clarity and color of the media after preparation.
• The final pH of the media.
• The presence of any particulates or precipitates after filtration.

Results:

The tissue culture media should be clear, of the correct pH, free of contaminants, and capable of supporting cell growth.

Precautions:

• Ensure all equipment and materials are sterile to prevent contamination.
• Avoid over-adjusting the pH, which can affect the stability of the media.
• Handle serum carefully to prevent contamination and degradation.

Conclusion:

The tissue culture media has been successfully prepared, sterilized, and supplemented, making it suitable for use in the culture of animal cells.