A penetration assay based on freeze-drying and vapour fixation was applied to show the spatial distribution of non-bound and bound cytostatic drugs in cellular spheroids. Several studies have proposed that peripheral binding of drugs correlates with limited penetration. We showed that granular accumulation, mainly at the peripheral part of spheroids, might occur in parallel with good penetration. For example, this was the case in human glioma spheroids after incubation with Adriamycin for 15-30 min. Following treatment with actinomycin D, colon carcinoma spheroids exhibited rather good penetration but also showed granular accumulation mainly in their peripheral regions. Ara-C accumulated largely and homogeneously in the peripheral regions of colon carcinoma spheroids and this severely delayed penetration. It took about 1 h for ara-C in the central regions of the spheroids to reach the same concentration as in the culture medium. In contrast, ara-C easily penetrated glioma spheroids without accumulating noticeably at the periphery. Retention tests involving washing and further incubation in drug-free culture medium revealed that the areas demonstrating extensive accumulation most often retained the drug, indicating binding, whereas the concentration of drug in other areas decreased. The oil-centrifugation method, which was used for rapid separation of the spheroids from the drug-containing medium, showed that the average concentration of daunomycin in the spheroids exceeded that in the culture medium as early as after 15 min, by which time only limited penetration had occurred. We found that good penetration of ara-C correlated with a low average concentration in glioma spheroids, whereas limited penetration correlated with a high average concentration in colon carcinoma spheroids. The latter finding was attributable to the high accumulation of drug at the spheroid periphery. Thus, there was an inverse relationship between penetration and binding and between penetration and average drug concentration. It seemed that binding delayed or prevented penetration, whereas little, if any binding resulted in better penetration. Granular binding such as that observed Adriamycin and actinomycin D gave intermediately good penetration.

The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogenous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.

The multicentre evaluation of in vitro cytotoxicity (MEIC) study is a programme designed to evaluate the relevance of in vitro toxicity tests for predicting human toxicity, and is organised by the Scandinavian Society for Cell Toxicology. The project started in 1989 and is scheduled to be finished by June 1996. MEIC is a voluntary effort by international laboratories to test the same 50 reference chemicals in their own in vitro toxicity systems. At present, 31 laboratories have submitted results for the first 30 reference chemicals from a total of 68 in vitro cytotoxicity tests. In the definitive evaluation of the MEIC programme, these in vitro results will be compared with human lethal blood concentrations and other relevant acute systemic toxicity data, and the results will be published as a series of articles. This paper, which is the first article in this series, describes and analyses the methodologies used in the 68 tests. The origins and purities of the test chemicals, the biological systems and the toxicity endpoints are also discussed. Since MEIC is not centrally directed, the selection of tests was entirely dependent on the preferences of the individual laboratories. Thus, the collection of tests is not representative of the full range of existing in vitro toxicity tests. In our study, basal cytotoxicity tests and ecotoxicological tests are prevalent, while tests for toxicity to primary cultures of differentiated cells, measured by organotypic toxicity endpoints, are clearly under-represented.