Passaging Hybridoma Cells: A Detailed Guide

Hybridoma cells are essential tools for producing monoclonal antibodies and are derived by fusing B lymphocytes (antibody-producing cells) with myeloma cells (cancer cells) to achieve both targeted antibody production and immortality. Properly maintaining and passaging hybridoma cells is key to their longevity and productivity in culture. This guide provides a step-by-step protocol and best practices for passaging hybridoma cells to ensure optimal health and antibody yield.

Materials and Equipment Needed

• Hybridoma culture medium: RPMI-1640 or DMEM, typically supplemented with 10-20% fetal bovine serum (FBS), L-glutamine, and antibiotics (e.g., penicillin-streptomycin).
• CO₂ Incubator: Set to 37°C with 5% CO₂ for optimal cell growth.
• Hemocytometer or automated cell counter: For determining cell concentration.
• Trypan blue or similar viability stain: To assess cell viability.
• Culture flasks: T-25 or T-75 flasks are common, but plates or bioreactors can be used depending on scale.
• Pipettes: For gentle handling of cells to avoid mechanical damage.

Preparing the Medium and Incubation Conditions

Hybridoma cells should be passaged approximately every 2-4 days depending on their growth rate. Optimal passage timing depends on:

• Cell Density: Passage when the culture reaches a density of 0.5 - 1 x 10⁶ cells/mL to prevent overgrowth and stress.
• Confluency: In suspension cultures, hybridomas may start to form clumps. Aim for a confluency around 70-80%.
• Nutrient Levels: As hybridomas proliferate, they consume nutrients rapidly, especially glucose
  and glutamine. Regularly check and replace the medium to maintain these levels.

Step 1: Check Cell Density and Viability

Assess Cell Density

• Use a hemocytometer or automated cell counter to assess cell density. An ideal range post-passage is between 0.2 - 0.5 x 10⁶ cells/mL, which allows space for growth.
• Calculate viable cell density using trypan blue staining to distinguish live cells (clear) from dead cells (blue). Aim for a viability of over 90%.

Assess Morphology and Aggregation

• Under the microscope, check that cells are rounded with a smooth membrane. Clumping or irregular shapes may indicate overgrowth or poor culture conditions.

Step 2: Dilute or Split Cells Based on Density

If the cell density exceeds the target range of 0.5 - 1 x 10⁶ cells/mL:

• Dilute the culture by removing part of the supernatant (spent medium) and replacing it with
  fresh, pre-warmed culture medium. This is essential to avoid nutrient depletion and maintain stable pH and osmolarity.

Alternatively:

• Split the culture by transferring part of the cell suspension to a new flask. For example, if the density is high, you might split 1:2 or 1:3, depending on the cell concentration and desired final volume.

Step 3: Resuspend and Transfer Cells

• Resuspend: Gently pipette up and down to create an even cell suspension.
• Transfer: Aliquot the required volume of cell suspension to the new flask containing fresh, pre-warmed medium.

Step 4: Incubate Under Optimal Conditions

• Place the flask in a CO₂ incubator at 37°C and 5% CO₂.
• Avoid disturbing the cells for at least 4-6 hours post-passage to allow them to acclimate and settle.

Prevent Overgrowth

Overgrown cultures suffer from nutrient depletion and can lead to decreased antibody production. Monitor cell density and passage cells before they exceed 1 x 10⁶ cells/mL.

Optimize Medium Composition

Hybridoma cells are highly metabolic and rapidly consume nutrients. Replace the medium frequently, and ensure adequate levels of glucose, L-glutamine, and serum for consistent growth. Consider supplementing with pyruvate if cells exhibit signs of stress.

Minimize Shear Stress

Hybridoma cells are sensitive to physical stress. Handle them gently during pipetting to avoid membrane damage. In some cases, spinning down cells at low speeds (around 200-300 x g for 5 minutes) can help if pelleting is necessary.

Monitor Contamination

Hybridoma cultures are vulnerable to contaminants, particularly mycoplasma, which can go undetected and alter cellular metabolism and antibody production. Test cultures routinely to ensure cell health and productivity.

• Use Conditioned Medium: For sensitive hybridomas, adding 10-20% conditioned medium from a healthy culture can improve viability and growth.
• Antibody Titer Monitoring: For hybridomas producing monoclonal antibodies, monitor antibody levels periodically using ELISA or Western blot to ensure consistent production.
• Optimize for Scale-Up: For large-scale antibody production, consider bioreactors or suspension flasks, and ensure consistent mixing and nutrient levels.

By following these detailed steps and best practices, you can successfully passage hybridoma cells to ensure their long-term viability, consistency, and productivity in generating monoclonal antibodies.