Dear scholars,
Our research is currently involved with hybridoma technology. We have isolated some original clones after fusion and we are preparing the subclone step to find some true positive clones. After subcloning by limited dilution (LD), we started to store the remaining original hybridomas into liquid nitrogen (2-5x106 cells/vial). After a month in liquid nitrogen, we cannot find any positive clones in the subclone stock. The supernatant we collected after thawing and culturing for 3 days from the original clone became negative compared to the supernatant before storing in liquid nitrogen. We tried to thaw and re-do the LD again but still have not found any positive clones.
+ We used PEG to induce fusion between myelomas and B-cells
+ We used indirect ELISA to screen for positive clones
+ We upscaled the positive original clone to 100mm culture dish (SPL Life Sciences) before subcloning by LD and store in liquid nitrogen.
+ We used NutriFreez® D10 Cryopreservation Medium to store hybridoma cells in liquid nitrogen (We stored the cells in a container box (Nalgene® Mr. Frosty) at -80oC at least 4 hours before moving them into -196oC in liquid nitrogen)
Has anyone encountered this situation before? Please let me know and I am very thankful for your answers.
Sincerely.
Are the cells still alive after thawing, i.e. do they divide? Maybe omitting the -80°C is worth a try, as ice crystals might form and destroy the cells. Try to thaw them quickly, e.g. by warming the vial in a water bath until you can throw the remaining "ice block" into a dish with medium.
Dear Dr. Wolfgang Schechinger,
Thank you for your answer. I would like to clarify some information:
1) Our hybridomas can live and divide normally after thawing (we've checked every 24 hours). However, like we mentioned above, it seems like our hybridomas "stop" producing antibodies. We tested this by ELISA.
2) We followed the protocol for cryopreservation of cells in DMSO. By reducing the temperature -1oC/min (using freezing container - Nalgene® Mr. Frosty).
We wonder if there are any cases which hybridomas lose the ability to produce antibodies after thawing from liquid nitrogen?
Hello, Cuong !
You see in this article, please;
Article Optimizing antibody production in batch hybridoma cell culture
Various models describing hybridoma cell growth and antibody production have been published in the last few years. Bree et al. (1988) predicted specific growth rate to be represented by a Monod relationship incorporating the growth-limiting medium components, glutamine, lactate, and ammonium. IgG production was described as being dependent on the glutamine concentration and the viable cell concentration. Equations representing glucose and glutamine utilization and ammonium and lactate production were also described. Miller et al. (1988) also introduced a Monod model for specific growth rate, but which incorporates the effects of glucase, glutamine, ammonium, and lactate concentrations. Similarly, Glacken et al. (1989) modeled specific growth rate in fed-batch culture as a function of serum activity and glutamine and ammonium levels.
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