Shortly after changing from submerged culture to ALI, holes have formed in the previously confluent cell layer. This is not due to mechanical damage and looks as though the cells are contracting away from each other which is letting basal media through the membrane and compounding the problem. This doesn't happen every time and our incubator set up is working properly. The cells aren't infected or unhealthy.
Hi Nadeene,
We have also seen this issue while culturing primary lens epithelial culture. Frankly speaking we were not able to understand the cause behind this observation but following little modifications in the culture protocol we were able to overcome this unwanted whole formation.
1. Try keeping seeding density ~15000/sq cm and always try to work with lower passage cells (p2-p3). Try determining the epithelial phenotye of the cells by immunocytochemical staining with E Cadherin, Cytokeratin 5/8 etc. If you see less expression of E Cad and more expression of N cad this is an indication of transformation of the epithelial cells to connective tissue type of messenchymal origin.
2. Do not culture cells for longer duration, that makes cell longer and retracting from each other. Try trypsinizing cells when 80% confluent. The wholes and retraction are common when the cells are confluent or over confluent.
3. Use appropriate cell culture supplements (EGF etc.) to keep cells healthy.
Though I must admit that the lens epithelial cells and the airway epithelial cells may not be behave similarly in culture and lens epithelium has an intrinsic property to form clusters of small colonies called lentoid (similar to spheroid) due to lens physiology and anatomy.
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