Methods and Principles from our Scientific Staff

Guidelines for sequential weaning of hybridoma cell lines from Classical Media with Fetal Bovine Serum (FBS) to AlphaGrow™ SF Hybridoma

  • Prior to initiating the weaning process, verify there is adequate stock of the frozen cell bank in the classical media with FBS.
  • For best results, cells should be freshly thawed and in log phase growth with >90% viability before starting the weaning process. The serum level for most hybridoma lines growing in classical medias (DMEM, RPMI, etc.) can typically be reduced to 5-10% within 1-3 passages post thaw.
  • While weaning hybridoma cells to AlphaGrow™ SF Hybridoma, the recommended seeding density is 2 x 105 viable cells/ml. 
  • Hybridoma cells should be passed every 1-3 days when viable cell count is 7 x 105 – 9 x 105 cells/ml. During the weaning process, viable cell count should not exceed 9 x 105 cells/ml. 
  • It is good practice to maintain a “back up flask” in the conditions of the previous passage.
  • Each cell line is different and exhibits unique growth characteristics and weaning tolerances. Cells should be monitored closely for changes in growth rate and/or viability. If changes are observed, continue passaging the cells in the current AlphaGrow™ SF/Classical Media ratios and serum level until growth rate and/or viability improve.

Easy Wean Protocol

  1. Begin the weaning process by passaging the cells in a 50/50 mix of AlphaGrow™ SF/Classical Media while maintaining serum level at 5-10% FBS.
  2. If growth and viability are good, passage cells in a 75/25 mix of AlphaGrow™ SF/Classical Media while reducing the serum level at 2.5-5% FBS (50% reduction).
  3. If growth and viability are good, passage cells in 100% AlphaGrow™ SF while reducing the serum level at 1.25-2.5% FBS (50% reduction).
  4. If growth and viability are good, continue to passage cells in 100% AlphaGrow™ SF while reducing the serum level to 1% FBS.
  5. If growth and viability are good, continue to passage cells in 100% AlphaGrow™ SF while reducing the serum level to 0.5% FBS.
  6. If growth and viability are good, continue to passage cells in 100% AlphaGrow™ SF while reducing the serum level to 0.3% FBS.
  7. If growth and viability are good, continue to passage cells in 100% AlphaGrow™ SF while reducing the serum level to 0.2% FBS.
  8. If growth and viability are good, continue to passage cells in 100% AlphaGrow™ SF while reducing the serum level to 0.1% FBS.
  9. If growth and viability are good, continue to passage cells in 100% AlphaGrow™ SF while reducing the serum level to 0.05% FBS.
  10. If growth and viability are good, continue to passage cells in 100% AlphaGrow™ SF while reducing the serum level to 0% FBS.
  11. If growth and viability are good, continue to passage cells in 100% AlphaGrow™ SF with the serum level at 0% FBS.

If a decrease in cell growth and/or viability is observed at any step, do not change the ratio of AlphaGrow™ SF/Classical Media  or decrease the serum level in the subsequent passage. Maintain the AlphaGrow™ SF/Classical Media ratio and serum level until growth and/or viability improve.

Certain cell lines may require additional supplementation during the weaning process. Try AlphaGrow™ SF Weaning Supplement (Prod. No.: S654) for weaning more sensitive cell lines. AlphaGrow™ SF Weaning Supplement protein free, serum free nutrient supplement can be used at concentrations up to 5%.

Example Weaning Schedule

Passage % AlphaGrow™
SF Hybridoma		 % Classical Media % Fetal Bovine Serum
1 50 50 5 – 10
2 75 25 2.5 – 5
3 100 0 1.25 – 2.5
4 100 0 1.0
5 100 0 0.5
6 100 0 0.3
7 100 0 0.2
8 100 0 0.1
9 100 0 0.05
10 100 0 0
11 100 0 0