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.
1. Preparing to Passage Hybridoma Cells
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
Ensure that the medium is pre-warmed to 37°C to reduce stress on the cells. Always work under aseptic conditions in a biosafety cabinet to prevent contamination, as hybridoma cells are particularly susceptible to microbial contaminants.
2. Understanding Passage Timing and Density
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.
3. Step-by-Step Protocol for Hybridoma Cell Passage
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.
4. Best Practices for Maintaining Hybridoma Cultures
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.
5. Troubleshooting Common Issues in Hybridoma Culture
Issue | Potential Cause | Solution |
---|---|---|
Low cell viability | Nutrient depletion or high passage density | Increase passage frequency, add fresh medium more frequently |
Decreased antibody production | Overgrowth or suboptimal medium composition | Maintain lower density, optimize serum and supplement levels |
Slow growth | Low serum concentration or contamination | Increase FBS concentration, test for contamination |
Excessive clumping | Cell aggregation at high densities | Gently pipette to dissociate clumps, passage more frequently |
High cell death post-passage | Mechanical stress during handling | Handle cells gently, avoid excessive pipetting |
6. Advanced Tips for Hybridoma Culture Maintenance
- 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.
Example Protocol for Hybridoma Passage
Step | Description |
---|---|
1. Determine Density | Count cells, aiming for 0.5 - 1 x 10⁶ cells/mL as a target density. |
2. Dilute or Split | If high, split 1:2 or 1:3; if within range, dilute to 0.2 x 10⁶ cells/mL. |
3. Resuspend and Transfer | Pipette gently to mix, and add to a new flask with fresh medium. |
4. Incubate | Place in CO₂ incubator at 37°C, 5% CO₂. |
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.
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