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

Issue title: Selected Presentations held at the 34th Conference of the German Society for Clinical Microcirculation and Hemorheology, Regensburg, Germany, 27–28 November, 2015

Guest editors: L. Prantl, E.M. Jung and F. Jung

Article type: Research Article

Authors: Krüger-Genge, A.^a | Fuhrmann, R.^b | Jung, F.^{a; *} | Franke, R.P.^b

Affiliations: [a] Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany | [b] Abteilung Biomaterialien, Zentralinstitut für Biomedizinische Technik, Universität Ulm, Albert-Einstein-Allee, Ulm, Germany

Correspondence: [*] Corresponding author: F. Jung, Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Kantstr. 55, 14513 Teltow, Germany. Tel.: +49 3328 352 269; Fax: +49 3328 352 452; E-mail: [email protected]

Abstract: BACKGROUND:The evaluation of the interaction of human, 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 bovine 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 phalloidin-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. Prostacyclin was analysed in the cell culture supernatant. RESULTS:1.5 min 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. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the supernatant increased significantly after 1.5 min to 466 ± 543 pg·mL–1 (p <  0.001) and after 5 min to 408 ± 458 pg·mL–1 (p <  0.001), while in control cells the prostacyclin 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 prostacyclin 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.

Keywords: HUVEC, morphology, microfilaments, adherence, culture medium

DOI: 10.3233/CH-151992

Journal: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 151-156, 2015