Desktop version

Home arrow Health arrow The Impact of Food Bioactives on Health

CONTENTS

Caco-2/Human Monocyte Derived DCs (Soluble Factors)

In comparison with the above mentioned system co-cultivation of cells in this way will only make it possible to study effect of communication caused by soluble factors. The epithelial cells and the DCs will not be able to be in direct contact with each other and communication can therefore only happen by soluble factors diffusing through the filter.

300 μL of a 1 × 106/mL Caco-2 cells suspended in RPMI 1640 with 10 % FCS, are seeded on filter inserts with 0.4 μm pores. The cells are maintained at 37 °C in 5 % CO2 atmosphere for 14 days. The medium in both compartments should be exchanged with fresh RPMI 1640 with 10 % FCS, every other day. At day 14, 1 × 106 CD14+ human monocytes in RPMI-1640 with 10 % FCS, 25 ng/mL IL-4 and 50 ng/mL GM-CSF are added to the lower chamber, facing the basolateral side of the epithelial monolayer. The medium in the lower chamber is exchanged with fresh RPMI 1640 with 10 % FCS, 25 ng/mL IL-4 and 50 ng/mL GM-CSF at day 4 of the co-cultivation. Maintain the co-culture for 6 days before experiments are conducted. Continue to exchange the medium in the upper chamber with fresh RPMI 1640 with 10 % FCS every second day.

The upper chamber will reflect the apical side and the lower chamber the basolateral side of the intestinal epithelium. Incubation time for the test compound will depend on type of compound and the read out system to be used and will have to be decided for each compound.

Caco-2/THP-1 (Soluble Factors)

300 μL of a 1 × 106/mL Caco-2 cells suspended in RPMI 1640 with 10 % FCS, are seeded on filter inserts with 0.4 μm pores. The cells are maintained at 37 °C in 5 % CO2 atmosphere for 21 days, exchanging the medium with fresh RPMI 1640 containing 10 % FSC every other day. At day 18 the THP-1 cells are plated at a density of 2 × 105 cells/mL in RPMI 1640 with 10 % FCS, 200 ng/mL IL-4, 100 ng/mL GM-CSF, 20 ng/mL TNF-α and 200 ng/mL ionomycin in a 24 well plate for 3 days. At day 21 the medium of the THP-1 cells is exchanged with RPMI 1640 with 10 % FCS and the filter inserts with polarized Caco-2 monolayers are placed into the wells together with the differentiated THP-1 cells.

The upper chamber will reflect the apical side and the lower chamber the basolateral side of the intestinal epithelium. Incubation time for the test compound will depend on type of compound and the read out system to be used and will have to be decided for each compound.

Caco-2/PBMCs (Soluble Factors)

300 μL of a 1 × 106/mL Caco-2 cells suspended in RPMI-1640 with 10 % FCS are seeded on filter inserts with 0.4 μm pores. The cells are maintained at 37 °C in 5 % CO2 atmosphere for 21 days with exchanges of the medium with fresh RPMI 1640 with 10 % FSC every other day. At day 20, PBMCs are plated in the basolateral compartment at a density of 1–2 × 106 cells/mL RPMI-1640 with 10 % FCS. PBMCs can be activated by anti-CD3/CD28, mitogenic lectins or LPS (see Chap. 15 PBMCs).

The upper chamber will reflect the apical side and the lower chamber the basolateral side of the intestinal epithelium. Incubation time for the test compound will depend on type of compound and the read out system to be used and will have to be decided for each compound.

Caco-2/B Cells

300 μL of a 1 × 106/mL Caco-2 cells suspended in RPMI-1640 with 10 % FCS are seeded on filter inserts with 3.0 μm pores. The inserts are placed in a 24 well plate with 1 mL of RPMI 1640 with 10 % FCS. The cells are maintained at 37 °C in 5 % CO2 atmosphere for 14 days with exchanges of the medium with fresh RPMI 1640 with 10 % FSC, every other day. At day 14, 1 × 106 Raji cells in RPMI 1640 with 10 % FCS are added to the lower chamber, facing the basolateral side of the epithelial monolayer. Maintain the co-culture for 6 days. Continue to exchange the medium in the upper chamber with fresh RPMI 1640 with 10 % FCS every second day.

The upper chamber will reflect the apical side and the lower chamber the basolateral side of the intestinal epithelium. To investigate uptake through M-cells the test compound should be added to the apical side. The incubation time for the test compound must be investigated different compounds, but are usually in the range of minutes to a few hours.

In all of the above-mentioned systems, the effects of test compounds on co-cultures should be compared with effects on matched filters with Caco-2 monocultures. This will make it possible to identify whether the effects of the test compounds are related to cell–cell communication in the co-culture systems.

 
Found a mistake? Please highlight the word and press Shift + Enter  
CONTENTS

Related topics