Determination of the cause of loss in Caco-2 cell

when Caco-2 cells were grown in the co-culture chamber. To test each hypothesis, a group of co-culture chamber wells were set up (as per section 2.4.2.2) while another group of wells were set up using an alternative method. TEER across the cell monolayers was measured and compared between the two groups. The various hypotheses, the methods used to test the hypotheses, as well as the outcomes from the experiments is summarised in Table 2.4.

One hypothesis was based on the fact that the basal compartment of the co- culture chamber is air-tight. Thus if pressure builds up within this compartment (e.g. due to the basal culture medium warming up from room temperature to 37°C), excess pressure has to be released via the microporous membrane, thus disrupting the Caco-2 cell monolayer. This hypothesis was tested by setting up the co-culture chamber without sealing the sampling port with the rubber septum, thus preventing any build up of pressure within the chamber well.

Another hypothesis was that the process of pushing the cell culture insert into the chamber lid, along with sealing the chamber well with the chamber lid, disrupts the cell monolayer. This was tested by removing the O-rings in the chamber lid to allow for a gentler movement when installing the lid and insert, thus reducing the risk of disruption to the cell monolayer. Furthermore, to test if the decrease in TEER was due to a combination of both of the above mentioned reasons, the co-culture chamber was set up with the O-rings removed, and with the sampling port left unsealed. However, as shown in Table 2.4, all of the above hypotheses were disproved.

An alternative hypothesis (Table 2.4) was that the basal side of the Caco-2 cells were not in contact with the culture medium. This may have been possible due to an insufficient volume of basal medium, or the formation of air bubbles beneath the microporous membrane. This hypothesis was tested by increasing the volume of basal medium such that the chamber well was filled to the rim, not allowing room for any air bubble formation. However, the extra medium pushed through the microporous membrane, abolishing monolayer integrity.

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Table 2.4 Summary of hypotheses developed to explain the observed loss of TJ integrity in Caco-2 cell monolayers when cultured in the co-culture chamber set up using the optimised method

Hypothesis Method for testing hypothesis Outcome 1 Pressure builds up within the chamber well, and escapes

through the microporous membrane disrupting the cell monolayer.

Did not seal sampling ports with rubber

septa. TEER decreased at a similar rate in both sealed and unsealed wells. 2 Basal side of the cell culture membrane is not in contact

with the cell culture medium (e.g. basal media volume is too low, air bubbles present underneath membrane).

Filled chamber well to rim with cell culture medium (no space for air bubble formation).

TEER plummeted as extra media pushed through membrane disrupting cell monolayer

3 The process of pushing the cell culture insert into the chamber lid and/or sealing the chamber well with the lid disrupts the cell monolayer.

Removed both outer and inner O-rings to allow for gentler movement when installing insert and lid.

TEER decreased at a similar rate in both wells with and without the O- rings.

4 A combination of hypotheses 1 & 3. Removed O-rings from chamber lid,

and did not seal sampling port with rubber septa.

TEER decreased at a similar rate in both wells with or without the O-rings and septa.

5 The top electrode plate isolates the Caco-2 cells from the

5% CO2 environment of the incubator. Did not assemble top electrode plate. TEER dropped by a similar amount in wells both covered and uncovered with the electrode plate.

6 Dark coloured build up on electrodes could be toxic to

It was also hypothesised that the top electrode plate of the co-culture chamber isolated the apical side of the of the Caco-2 cell monolayer from the atmosphere of the incubator, implying that the Caco-2 cells may be exposed to a non-favourable environment on the apical side. However the TEER across Caco-2 cell monolayers dropped by a similar amount in wells both covered or uncovered by the electrode plate.

It was then hypothesised (Table 2.4) that the observed phenomenon may be inherent to the co-culture chamber and not necessarily due to the method of chamber assembly. It was noted that a dark coloured residue had built up on each of the electrodes, and it was hypothesised that this residue may be toxic to the Caco-2 cells. To test this hypothesis, electrodes were cleaned with metal polish (Autosol). However the TEER dropped faster in Caco-2 cells grown in wells where the electrodes had been cleaned, and the residue had begun to build up again following the incubation period. Nonetheless this did not disprove that the exposure to the electrodes had an adverse effect on the Caco-2 cells.