Growth surface-induced gene and protein expression patterns in Caco-2 cells.

The underlying matrix plays an important role in the adhesion, proliferation and differentiation processes of Caco-2 cells. When culturing these cells for pharmaceutical purposes it is essential to know the influence of different supports on morphological and functional cell parameters. The impact of polystyrene, Matrigel-coated polystyrene, glass and nanostructured Easy-To-Clean (ETC01) slides was investigated over time by real-time quantitative reverse transcription polymerase chain reaction, enzymatic assays and immunofluorescent staining techniques. Compared to polystyrene, ETC01 slides induced cellular activities towards functional differentiation after short cultivation times. Glass significantly accelerated the differentiation process up to day 10 in culture, while Matrigel-coating had no significant benefit. By day 21 postseeding, the phenotype had equalized as indicated by constant brush border enzyme activity and villin mRNA expression masking the initial differences between the supports. The accelerated differentiation on specific matrices could be advantageous as it may enable cultured monolayers to be used earlier, and has to be considered when interpreting and comparing results.

Validation of reference genes for qPCR studies on Caco-2 cell differentiation.

Validation of reference gene expression stabilities is a prerequisite for reliable normalization of qPCR data. The present study assessed the variation of six reference genes (ACTB, GAPDH, B2M, HPRT1, SDHA, YWHAZ) in Caco-2 cells under the influence of different growth supports and cultivation periods. Genes were ranked according to their stability using the geNorm software. To verify the influence of reference gene selection, ALPI gene expression during differentiation was quantified using the most or the least stable reference gene for normalization. Experimental conditions significantly affected the expression levels of reference genes. Whereas GAPDH and ACTB were revealed as most stable genes, SDHA was the least stable one. The extent of ALPI gene expression was significantly changed by the selection of the reference gene. This study provides a basis for qPCR studies related to both the differentiation process of Caco-2 cells and the elucidation of cell behaviour influenced by surface modifications.

Influence of surface modification on vitality and differentiation of Caco-2 cells.

It is widely accepted that the functional and morphological differentiation of cells is initiated and determined by the interaction of molecules of the extracellular matrix and adhesion molecules of the cell membrane. To assess the influence of the underlying matrix on the characteristics of cells, enterocyte-like Caco-2 cells were cultivated on substrates commonly used for cell culture as well as on glass coated with hydrophobic layers. Providing the same starting conditions for growth, the parameters investigated on preconfluent Caco-2 cells were the number of adhering cells, the proliferative activity and the degree of differentiation indicated by the expression of three brush border enzymes. Whereas tissue culture treated polystyrene elicited highest rates of adhesion, proliferation, and differentiation, even glass altered the pattern of brush border enzyme expression. The hydrophobic surfaces strongly decreased the adhesion and the proliferation but the surviving cells exhibited a pronounced higher degree of differentiation. Interestingly, each sub-type of hydrophobic matrix triggered a different pattern of brush border enzyme expression. Thus, the development of a certain phenotype of a cell can not only be triggered by certain components of the extracellular matrix but also by artificially prepared surface coatings of the underlying matrix. In the future it seems to be feasible that cells can be programmed by tailoring the surface of the underlying substrate.

Selection of reliable reference genes for qPCR studies on chondroprotective action.

BACKGROUND: Chondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected step in the analysis process. Therefore, the present study aimed to determine the expression stability of common reference genes (ACTB, Beta actin; GAPDH, Glyceraldehyde-3-phosphate; B2M, Beta-2-microglobulin; HPRT1, Hypoxanthine phosphoribosyl-transferase I; SDHA, Succinate dehydrogenase complex, subunit A; YWHAZ, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) under the influence of glucosamine, curcumin and diacerein in the IL-1beta-stimulated C-28/I2 chondrocyte model, using the geNorm software tool. RESULTS: CPA treatment of C-28/I2 chondrocytes significantly affected the expression level of many reference genes (p < 0.05). According to their expression stability, geNorm analysis revealed rankings of the 3 most stable genes (from most stable to least stable) as follows: GAPDH, B2M and SDHA in glucosamine treated samples and HPRT1, GAPDH and B2M in curcumin or diacerein treated samples. Interestingly, ACTB was one of the most variably expressed genes throughout all experiments. CONCLUSION: Our study points out the problem of relying on commonly used reference genes without an accurate validation process. For normalization purposes in gene profiling studies on glucosamine action, the genes GAPDH, B2M and SDHA are recommended as single reference genes depending on the expression level of the target gene or more favourably in combination. For experiments with curcumin and diacerein the use of HPRT1, GAPDH and B2M should be considered.