Permanent cell line expressing human factor VIII-related antigen established by hybridization.

Abstract

A permanent human cell line, EA hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell, line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included, a marker. chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIH-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA-hy 926 represents a permanent line.Differentiated functions of endothelium are critical for the vascularization process in normal and neoplastic tissue, for maintaining the blood-brain barrier, and for hemostasis. Factor VIIIrelated antigen (VIIIR:Ag) is an endothelial cell product (1) involved in the aggregation of platelets, and megakaryocytes are the only other cell type known to express this antigen (2).VIIIR:Ag is present in normal human plasma at about 10 pug/ ml, and decreased levels are found in classical von Willebrand disease, an autosomal dominantly inherited bleeding disorder in humans. VIIIR:Ag circulates as a large molecular complex and its platelet-aggregating activity is directly related to the size distribution of the complex (3). Factor VIII coagulant activity (antihemophilic factor) is also associated with the VIIIR:Ag complex in plasma. We have previously described interspecies hybrids between human endothelial cells and a number of rodent cell lines, in which VIIR:Ag was not expressed (7). There has also been a preliminary report by others (8) of human-rodent hybrids that may express VIIIR:Ag, but the intracellular distribution of antigenicity that they detect is not similar to that of VIIIR:Ag in endothelial cells.Here we report a human intraspecies hybrid, EA-hy 926, that expresses VIIIR:Ag with the same morphological distribution as in primary endothelial cells. MATERIALS AND METHODSCell Culture. Cells were cultured on plastic ware at 370C in a humid atmosphere containing 7% CO2 in air. Culture medium was exchanged every 3-5 days. At confluence, 0.01% trypsin (TRL Worthington, no. LSOO 044 52) in 8 mM phosphate-buffered saline (pH 7.4) with 0.54 mM EDTA and 5.5 mM glucose was used to detach the cells, which were then subcultured at lower cell density.Human umbilical vein endothelial cells (HUV-EC) were isolated as described in detail by Gimbrone (9). The vein of an umbilical cord kept at 40C for 4 hr postpartum was irrigated with phosphate-buffered saline. The endothelial cells were dissociated from the vessel wall with 0.1% collagenase (Sigma, no. C2139) in phosphate-buffered saline at 370C for 20 min. The cells were separated from the collagenase solution by centrifugation and were distributed over...

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