Development of Assay Ready Frozen Cells for a rapid permeability Assay

Abstract

Caco-2 and MDCK are widely used as in vitro-models in the pharmaceutical industry to predict the abortion of new drug candidates in the early stage of drug development. Conventional transport studies in human colon carcinoma cell line Caco-2 are carried out after a culture period of 21 days on a filter support therefore the cells have to be continuous maintained. in parallel to drug screening activities. To save time and cost test compounds can tested already on a 4-day old monolayer. The use of frozen cells has furthermore the advantage, that cells are always ready for seeding into plates. The aim of this thesis is to develop Assay Ready Frozen Cells for a rapid permeability Assay. MDCK as faster growing alternative to Caco-2 cells, show a less tight monolayer, with a maximal TEER value of 30.7 Ω∙cm². Two freezing media are used to cryopreserve cells, the “standard freezing medium” and the “New Freezing Medium Generation 2” (NFM-G2). The freezing and thawing process delays the cell proliferation shown by growth curves in comparison to fresh cells. Caco-2 viability after thawing and cultured is higher in NFM-G2. The marker molecule Texas Red Dextran was used to investigate the monolayer integrity within 4 days. Cells frozen in both media show equivalent results in the permeability assay. Fresh Caco-2 cells, show a permeability value of 0.4%. The permeability value of frozen cell monolayers is 0.8%. Subclones are isolated from parenteral cell line, differ in P-gp expression. On an optimized protocol for the permeability assay is was able to obtain. The P-gp transporter serves as indicator for the functionality of the monolayer. TEER values in subclones were already high after the second day. The values were in the range of at least 200 Ω∙cm². The highest TEER value is 1193.5 Ω∙cm². It is measured in one sample of the frozen, parenteral cell line at day 6. Papp values which were obtained with continuous cells grown for 20 to 24, were achieved, with frozen cells within 7 days. Subclones are frozen with three freezing devices. In this work Mr. Frosty shows the best results, but differs only marginal from the other devices The subclone D5 shows a good viability before and after freezing, independent of the freezing medium and device In the end the results show, that Caco-2 cells can be used for the intestinal permeability assay as frozen cells, if the cryostocks are prepared from cultures of optimal quality and by the use of NFM-G2 in combination with a controlled rate freezer. Sub-cloning changes the properties of the cell lines, indicating that commercially available Caco-2 and MDCK cell lines are heterogeneous cell populations. The results obtained with the isolated subclone D5 are close to the reference values. There is no need to switch to a new clone. Nevertheless, subclone F9 shows no P-gp activity in the calcein AM assay. Here a Caco-2 clone is available which might be interesting if a P-gp activity of Caco-2 cells is unwanted, e.g. as basic cell line for the expression of genetically modified P-gp.