Morphology of primary XXXX venous endothelial cell cultures before and after culture medium exchange.

@entity1 @entity291 @entity6286 @entity4770
BACKGROUND: The evaluation of the interaction of @entity1 , venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. MATERIAL AND METHODS: HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from @entity291 corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with @entity6286 -rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. @entity4770 was analysed in the cell culture supernatant. RESULTS: 1.5min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The @entity4770 concentration in the supernatant increased significantly after 1.5min to 466    543 pg  mL-1 (p< 0.001) and after 5min to 408    458 pg  mL-1 (p< 0.001), while in control cells the @entity4770 concentration did not change remaining in the range of 50    48.9 pg  mL-1. CONCLUSION: This study revealed that the exchange of the cell culture medium led to a rapid disturbance of the HUVEC with stress fiber formation, disconnection of cell-cell contacts and an altered @entity4770 secretion, which had regressed nearly completely after 12 hours. Therefore, the evaluation of HUVEC on body foreign materials should be performed not earlier than 12 hours after cell culture medium exchange to avoid a misinterpretation of the endothelial cell morphological state. This procedure minimizes the risk of a misinterpretation of the endothelial cell morphology - caused by the culture medium exchange and not by the interaction between biomaterials and HUVEC.
