Important confrontation is to develop on in vitro assay system for detection of metabolic activation of various immunosuppressive agents including cyclophosphamide(CP) on lymphocyte cultures. Limitation, however is that lymphocyte is incapable to metabolize the agents. To overcome this, the cocultivation method of mouse lymphocyte with rat hepatocyte which are capable of metabolic activation of the agents was developed in this study. Three splenic lymphocyte cultures were used ; Mishell-Dutton assay for in vitro antibody formation, splenic lymphocyte responsiveness to mitogens and natural killer(NK) cell assays.
Lymphocyte in fluid medium were placed over soft agarose matrix containing rat hepatocyte layers within 60×15 mm culture dishes and treated simultaneously with CP. Following incubation for 2hr in 5% $Co_2/95%$ air 37℃ humidified incubator, lymphocytes were harvested, washed and assayed by above three parameters. There was little effect of CP at doses up to 1mM on any of the parameters measured, unless activated by lymphocytes.
When splenic lymphocytes were exposed to CP in the presence of hepatocytes, a dose related inhibition of plaque forming cells (PFC) in the Mishell-Dutton assay, lymphocyte proliferation to mitogens measured by 3H-Thymidine uptake and NK cell activity of target cell lysis, were observed. Thus results suggest that hepatocyte-lymphocyte cocultivation system is well suited for detecting the immunosuppressive agents which require metabolic activation.