Study of Growth Factors in Spent Medium for Better Mammalian Cell Culture
Monoclonal antibodies are widely used as anti-cancer drugs, extracorporeal diagnostic agents, etc. They are produced by mammalian cells such as hybridoma and CHO cells. Generally, these cells need growth factors such as insulin and transferrin in order to enhance the cell proliferation and antibody productivity therefore culture of antibody producing cells is very costly. In this study, we tried to discover useful materials to enhance antibody production and we focused on spent medium after antibody purification. In the spent medium, there were several proteins produced by antibody producing cells. It was cut off low-molecular compounds and concentrated by ultrafiltration module then the concentrated solution was named spent medium derived supplement. Spent medium derived supplement enhanced the cell proliferation and antibody production significantly when it was used with insulin and transferrin. These results indicate that spent medium after antibody purification has some attractive proteins or peptides which enhance cellar functions of antibody producing cells synergistically by combination of insulin and transferrin.
KeywordsAntibody Production DMEM Medium Human Antibody Spend Medium Trypan Blue Exclusion Assay
1 Materials and Methods
Cell line and culture condition: CHO-DP12 (ATCC, USA) was used as a model of antibody producer. It produces recombinant human anti-interleukin-8. It was usually cultured in DMEM medium (Nissui, Japan) supplemented with 5% (v/v) FBS, 0.2% sodium bicarbonate, 10 mM HEPES and 2 mM glutamine, at 36.5 Celsius degrees in humidified air containing CO2 at 5%.
Antibody production and purification: CHO-DP12 cells were seeded on a 90 mm culture dishes (Sumitomo Bakelite, Japan) in DMEM medium containing 5% FBS. The next day, adhesion to the dish was confirmed then the medium was removed. The cells were washed with PBS four times in order to remove the FBS completely. A serum-free medium ASF104 (Ajinomoto, Japan) containing insulin (5 mg/l) and transferrin (5 mg/l) was added to the dishes. These cells were cultured for more 3 days. The culture supernatant was collected at the end of the culture period.
Purification of antibody and treatment of spent medium derived supplement: The collected culture supernatant was loaded into affinity culumn (GE Healthcare, UK) to separate antibody and the flow-through fraction. The flow-through solution was collected then concentrated by ultrafiltration unit (Millipore, USA).
Proliferation assay and measurement of antibody production: CHO-DP12 cells were seeded on 24-well culture plates (Sumitomo Bakelite, Japan) in DMEM medium containing 5% FBS at 16000 cells/well. The next day, adhesion to the bottom of the well was confirmed then the medium was removed. Each well was washed with PBS four times. After that, ASF104 containing supplements, insulin (5 mg/l), transferrin (5 mg/l), and/or spent medium derived supplement, was added to the each well by 500 μl. The cells were cultured for more 78 h. The culture supernatants were collected at the end of the culture period. The viable cell density and viability were determined by counting in a hemacytometer under a phase contrast microscope using the trypan blue exclusion assay. The amount of recombinant human antibody in the culture supernatant was determined with sandwich enzyme-linked immunosorbent assay (ELISA). For the ELISA, the capture antibody was rabbit affinity purified antibody to human IgG (Bethyl Laboratories, USA), the secondary one was goat antibody to human IgG (γ) conjugated with horseradish peroxidase (American Qualex Antibodies, USA). o-phenylenediamine dihydrochloride (Nacalai Tesque, Japan) was used as the substrate. Human antibody concentration was calculated from optical density at 490 nm (Thermo Fisher Scientific, USA).
2 Results and Discussions
In conclusion, we studied the effect of spent medium derived supplement on proliferation and antibody production of antibody producer. The spent medium derived supplement improved the proliferation and antibody production under action of insulin and transferrin.
- A. Ogawa, S. Fukui, S. Terada, Enhancement of antibody production using solution after antibody purification. Animal Cell Technology: Basic & Applied Aspects, 16, 145–150 (2010)Google Scholar