Caco-2 cells are routinely maintained in RPMI 1640 (alternatively Dulbecco’s Modified Eagle Medium, DMEM) with 10 % fetal bovine serum and the following additions: 1 % non-essential amino acids (NEAA), 50 μM thioglycerol, 25 mg/ml gentamycin (complete medium). The cells should be kept at 37 °C in a humidified atmosphere containing 5 % CO2. For propagation in culture flasks, Caco-2 cells are seeded in a concentration of 105 cells/cm2. Medium should be changed every 3 days. At 80 % confluence, typically after 4–5 days, the cells are split 1:10 before further cultivation. Trypsinize Caco-2 cells by first rinsing with PBS (15 ml pr. 75 cm2 flask). Add 5 ml trypsin/EDTA solution, rinse the cells and pour off most of the trypsin/EDTA. Only 1 ml trypsin/EDTA solution is necessary to wet the whole cell layer in a 75 cm2 flask. Incubate the flask at 37 °C for 6–15 min. The incubation with trypsin should be as short as possible as this process will affect cell viability. As soon as the cells are detached, stop trypsinization by adding complete medium containing fetal calf serum. Transfer the cells to a test tube and let cell aggregates and debris sediment. Transfer the supernatant to a new test tube, and take an aliquot to count the cells. Check cell viability. The content of dead cells should not exceed 5 %.
Protocol for Polarizing Caco-2 Cells in Tissue Culture Inserts
Caco-2 cells can be cultured on a filter support. The filter support can be made from polycarbonate, polyester or polyethylene terephthalate. Particularly the latter is claimed to be inert with low non-specific protein-binding properties. The filter supports can be transparent or translucent. If you want to follow the differentiation process in the inverted microscope and prepare the differentiated cells for scanning electron microscopy, transparent filters are preferred. Furthermore, the filters can come with different pore sizes ranging from 0.4 to 8 μm. To study parameters like transcellular transport and permeability, 0.4 μm filters are recommended. Insert with larger pore sizes can be exploited if direct cell–cell interactions are being scrutinized, i.e. in co-cultures of Caco-2 cells with stromal cells or adherent immune cells.
Depending on inherent properties of the particular Caco-2 cells available, it may be necessary to coat the filters with protein to prevent the cells from detaching in the final stages of the differentiation process. Some vendors offer ready-coated filters. However, efficient coating of the filters can be carried out by covering them with collagen solution Type I from Sigma (1/100 in water). Incubate at room temperature for 3–4 h. Remove the collagen solution and leave the filter inserts to dry overnight.
Place the necessary number of filter inserts in a 12 well plate. Dilute the cell suspension to a concentration of 1 × 106/ml. Seed 0.5 ml of the cell suspension to each insert corresponding to seeding density of 500,000 cells pr. 12 mm filter insert.
This seeding density corresponds to 4 × 105 cells/cm2. Add 1.5 ml medium to the basolateral compartment in a 12 well plate, and 0.5 ml to the apical compartment. Depending on experience it is recommended to start with setting up filters in quadruplicate, that is later reduced to triplicate or even duplicate. Medium should be changed on days 4, 8, 12, 16 and 18. Cells will be fully polarized by day 21.
The differentiation of Caco-2 cells seem to follow a time schedule in the expression of morphological and biochemical properties of the absorptive enterocytes. Due to the cellular heterogeneity of the cell line the differentiation process occurs in a mosaic pattern, with some areas expressing fully differentiated cells with microvilli after 12–14 days while other areas contain less differentiated cells. According to the protocol above, the Caco-2 monolayer will be homogenously differentiated after 18–21 days. When polarized and confluent, the cell layer forms a continuous barrier between the upper and lower compartments (apical/mucosal and basolateral/serosal). The compound of interest, suspended in a physiological fluid, typically PBS, is added to the upper compartment, and the increase in concentration in the lower compartment is measured. In this way the permeability, absorption and transepithelial transport of any nutritional compound can be studied. Special emphasis should be paid to compounds or drugs that are poorly soluble in physiological buffers. If stock solutions of the compound of interest has to be made in organic solvents like ethanol or dimethylsulfoxide (DMSO), be aware that organic solvents may affect the cell layer in various ways. Always include a filter/filters for solvent control. In general, avoid using higher solvent concentrations than 1 % (v/v) or else.