SYNOPSISA novel artificial extracellular matrix derivatized with a cell adhesive peptide, Arg-GlyAsp-Ser (RGDS) tetrapeptide, which is the central peptidyl sequence of the adhesive site of fibronectin, was designed. RGDS coupling was achieved via isocyanation of surface hydroxyl groups of poly (vinyl alcohol) (PVA) film and subsequent conversion to activated ester. The surface-modified PVA film was quantitatively analyzed by ESCA. The surface density of RGDS was partly controlled by the degree of isocyanation of the PVA film. Bovine endothelial cells ( ECs) adhered and grew well on the RGDS-derivatized PVA film, irrespective of the presence or absence of the serum. The adhesion and growth of ECs were enhanced with an increase in the surface density of RGDS. When a sufficient amount of RGDS was added to the medium, the adhered ECs were delaminated. This indicated that the adhesion of ECs on an RGDS-derivatized PVA surface is mediated by the RGD-ligandl receptor interaction. Thus, a peptidyl artificial matrix via surface derivatization was developed. 0 1993 John Wiley & Sons, Inc. I NTRODU CTlO NLiving cells in the tissues adhere to and grow on extracellular matrices ( ECMs ) that are composed mainly of proteins and glycosaminoglycans. It has been established that the ECMs regulate cell behavior such as adhesion, growth, migration, and differentiation. Of biomolecular components of ECMs, it has been reported that adhesive proteins such as fibronectin, collagen, vitronectin, and laminin serve as a cell-adhesive matrix. It has been established that the minimal amino acid sequence of the adhesive site of fibronectin is Arg-Gly-Asp-Ser (RGDS; one-letter description of amino acid) .1,2 In fact, previous studies showed that peptides containing the RGD sequence dose-dependently inhibited the cell-adhesion promoting effect of fibronectin when mixed with various anchorage-dependent cells The background, as mentioned above, on the celladhesion mechanism via RGD-ligand/ receptor interaction prompted us to design an artificial extracellular matrix into which an RGD-ligand is incorporated. Imanishi et al. reported that the RGDS tetrapeptide immobilized on polyamine-grafted silicone films significantly enhanced the adhesion of fibrob l a s t~.~ We have attempted to prepare RGD-derivatized surfaces and As a part of the series of our RGD-peptidyl-immobilized studies aiming at the development of 2-dimensional and 3-dimensional artificial ECMs, a surface derivatization approach is demonstrated here.In this paper, we report a surface derivatization of RGDS on a nonadherent hydrophilic surface, poly (vinyl alcohol) (PVA) . RGDS derivatization resulted in the conversion of the surface from nonbioactive to bioactive. 1983
Tyrosine aminotransferase (TAT) induction and albumin secretion abilities were examined in rat hepatocytes immobilized within calcium alginate; the immobilized hepatocytes lost these abilities within a week. An attempt was then made to immobilize multicellular spheroids of hepatocytes for the purpose of stabilizing the liver functions. Although it takes at least 4 days to form spheroids in the conventional method using monolayer‐cultured cells, in this study we developed a new method for rapid spheroid formation. Isolated hepatocytes were seeded into a polystyrene dish and incubated on a rotary shaker. Hepatocytes started to aggregate after 6 h of the rotation culture, and spheroids approximately 100 μm in diameter formed within 24 h. The immobilized spheroids had higher TAT induction and albumin secretion abilities, which were maintained for a longer time, than the immobilized nonaggregated cells. Further stabilization was observed in immobilized heterospheroids formed in the presence of non‐parenchymal liver cells. This method for the rapid formation of spheroids consisting of hepatocytes and non‐parenchymal liver cells could be utilized in the construction of a bioartificial liver support system.
Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.
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