Splenocyte culture - Cell death upon in vitro culture (Apr/26/2006 )

Dear all,

I am trying to develop a protocol for isolation of splenocyte from immunized rat spleen, culture cells in vitro for stimulation of proliferation by known mitogen (con A) and use these primed cells for transfer of disease into healthy rats (adoptive transfer of disease).

I isolate cells using ficoll (Amersham) from homogenised spleen (dounce homogenisation to make single cell suspension). Cell yield is about 2 x 10exp7 cells/spleen of healthy rat. I am using healthy rat to optimise protocol. Splenocytes are cultured in RPMI + 2mM glutamine + 4.5g/L glucose + 10mM HEPES + 1mM Sod.pyruvate + 1.5g/L Sod bicarbonate + 0.05mM 2 ME + 10% FCS + antibiotics(pen-strep) + ConA 50ug/ml.

After in vitro culture for 4 hours, significant population (~40%) adhere to the flask, only 30% cells remain viable, remaining non viable.

After 24-48 hrs, barely 5-10% population is viable.

I am wondering why cells are not proliferating. Any suggestions please?

I think macrophages adhere to the falsk. I don't think they proliferate. They are primary cells.
why you don't isolate them and negatively or positively select the cell population you are interested in and use it directly without culturing.

If you succeed in your culture please let me know! I have a lot of samples and I prefere if I can culture or freeze them rather than working for 24 hours.


Good luck