Anmeldung zur Tagung
Registration
Grußwort
Invitation
Themen
Topics
Allgemeiner Ablauf
General overview
Wissenschaftliches Programm
Scientific program
Kurse
Courses
Symposien
Symposiums
Frühstück mit Spezialisten
Breakfast with specialists
Arzthelferinnen-Fortbildung
Rahmenprogramm
Social program
DOG Information
DOG Information
Allgemeine Informationen
General Information
Autorenindex
Index of Authors
Ausstellerliste
Exhibitors
Sponsoren
Sponsors
Teilnahmegebühren
Registration fees
Impressum
Influence of cell culture conditions on phagocytosis of photoreceptor outer segments (POS) by SV40-transfected retinal pigment epithelium
Karl M., Valtink M., Bednarz J., Engelmann K.
Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Augenheilkunde, Martinistr. 52, 20246 Hamburg
Objective: Loss of epithelial characteristics and specific functions
during in vitro cultivation may contribute to unsuccessful transplantation
of RPE (and other) cells. We present a method to determine the phagocytic
capacity of SV40-transfected RPE cells (SV40-RPE) in dependence upon culture
conditions.
Methods: SV40-RPE were seeded in 96-well plates with a defined
number of cells. Before confluence, cells were cultured serum free for
12h and subsequently were incubated with the test media for another 12h.
This study examines MEM(E), DMEM (low glucose), Medium F99 (Medium 199/Ham´s
F12) and a commercially available serum free medium, the latter two media
supplemented with serum at concentrations ranging from 0-15%. Cells were
incubated with SNAFL®-2 labelled POS and binding and indigestion were
measured after 4h using a cytofluorometer.
Results: The established assay allows simultaneous quantification
of binding and indigestion of POS. Phagocytosis was characterized in dependence
on POS-concentration, incubation duration, POS-specifity (labelled vs.
unlabelled), medium composition and serum supplementation. Basically,
increase of serum up to 10% led to an increase in phagocytic rate, whereas
with higher serum concentrations phagocytic rate declined. The influence
of basal medium composition became visible only when supplemented with
serum. While highest phagocytic rate in serum -free medium could be achieved
with 10% serum supplementation, a comparable level could be achieved in
medium F99 with only 5% serum supplementation. It appeared that proliferation-promoting
media had only a moderate influence on stimulation of phagocytosis.
Discussion: Correlation of cell function and morphology in vitro
is up to now only poorly understood. With the presented method, phagocytosis
as a specific function of RPE cells can be determined in dependence upon
culture conditions. Supported by: Werner-Otto Stiftung