Immobilization of ligand-modified polyamidoamine dendrimer for cultivation of hepatoma cells.

Cationic polyamidoamine dendrimers are known to be highly branched cascade polymers. The core part of these polymers, tris(2-aminoethyl)amine, was immobilized onto polystyrene plates to which animal cells do not adhere. using photoreactive 4-(3-trifluoromethylazirino) benzoyl-N-succinimide (TDBA-OSu). Cells of a rat hepatoma cell line, H4-II-E-C3, adhered to a surface immobilized with a first-generation dendrimer probably through interactions between the terminal amino groups of the dendrimer and the cell membranes. The adhered cells were viable, could proliferate, and exhibited urea synthetic activity. The modification of the terminal amino groups with fructose increased the final number of cells obtained after 5 days of cultivation. Multigeneration dendrimers were prepared by repeated linkage of tris(2-aminoethyl)amine with the amino groups. Theoretically, the number of terminal amino groups available for ligand modification is twice as much for each generation of dendrimer growth. Cells cultivated on multigeneration fructose-modified dendrimers exhibited enhanced urea synthetic activity. The use of ligand-modified dendrimers is, therefore, considered to be very promising for the construction of bioartificial organs based on cultivation of the animal cells.

Immobilization of tripeptide growth factor glycyl-L-histidyl-L-lysine on poly(vinylalcohol)-quarternized stilbazole (PVA-SbQ) and its use as a ligand for hepatocyte attachment.

A tripeptide growth factor, glycyl-L-histidyl-L-lysine (GHK), was immobilized on the surface of poly(vinylalcohol)-quarternized stilbazole (PVA-SbQ) gel. The photoreactive substance, 4-(3-trifluoromethylazirino)benzoyl-N-hydroxysuccinimide (TDBA-OSu), was employed to link the gel and ligand GHK. The density of immobilized GHK was 70 nmol/cm2. Isolated rat hepatocytes were inoculated on the GHK-immobilized PVA-SbQ gel and cultured for 5 d. About 24 h after inoculation, hepatocytes started to aggregate and formed multicellular spheroids while almost no cells attached to GHK-non-immobilized PVA-SbQ gel. The formed spheroids attached firmly to the surface of PVA-SbQ gel for 5 d. GHK was, thus, shown to be an effective ligand for hepatocyte attachment. Dodecamethylenediamine was used to extend the length between the gel surface and GHK. Extension of the length significantly increased the number of attached hepatocytes.

Effectiveness of polyamidoamine dendrimers modified with tripeptide growth factor, glycyl-L-histidyl-L-lysine, for enhancement of function of hepatoma cells.

Cationic polyamidoamine dendrimers are known to be highly branched cascade polymers. Tripeptide growth factor, glycyl-L-histidyl-L-lysine (GHK), was employed as a ligand for activation or attachment of cells from a rat hepatoma cell line, H4-H-E-C3, and immobilized at the terminus of the dendrimer (GHK-dendrimer) to develop a suitable surface for use as a culture substratum in the bioartificial liver support system (BAL). The growth of cells was inhibited by increasing the number of generations of GHK-dendrimers. On the other hand, urea synthesis and lidocaine clearance of the cells adhered on fifth generation GHK-dendrimers were enhanced much more than on first generation GHK-dendrimers. GHK was shown to act as a growth inhibitor and an activator of hepatoma cells. These properties of GHK are advantageous for the utilization of hepatoma cells in BAL. Ligand-modified dendrimers are very promising for the creation of a high-performance substratum for cell culture and high performance bioartificial organs, as well as for high-performance bioartificial liver systems. GHK may have the potential to be a highly useful ligand.

Application of glutaraldehyde-crosslinked chitosan as a scaffold for hepatocyte attachment.

The effectiveness of chitosan, a biocompatible polymer derived by the deacetylation of chitin, as a scaffold of hepatocyte attachment, was examined. Since chitosan gel was too fragile to use for cell culture, its free amino groups were crosslinked by glutaraldehyde to increase its strength. Rat hepatocytes seeded onto glutaraldehyde-crosslinked chitosan (GA-chitosan) gel could stably attach to the surface, retaining its spherical form, the same as in vivo, and then release a very small amount of lactate dehydrogenase during the 5 d culture period. By contrast, hepatocytes on a collagen-coated surface spread flat, and they released much more lactate dehydrogenase than those on the GA-chitosan gel. Hepatocytes on GA-chitosan also retained higher urea synthesis activity, a liver-specific function, than those on the collagen-coated surface. These results indicate that chitosan is a promising biopolymer as a scaffold of hepatocyte attachment, which can be applied to an effective bioartificial liver support system.

Effectiveness of fructose-modified chitosan as a scaffold for hepatocyte attachment.

Free amino groups of chitosan, a substance which has previously been shown to be a good scaffold for hepatocyte attachment, were covalently modified with fructose. The modification significantly increased the number of cells that could be attached on the surface of chitosan gel. Rat hepatocytes cultivated on fructose-chitosan behaved similarly to those on unmodified chitosan, i.e., they retained the spherical shape they have in vivo, and released much less lactate dehydrogenase than cells attached on a collagen-coated surface. The modification with fructose did not alter the important characteristics of chitosan for hepatocyte culture: liver-specific functions such as urea synthesis and drug metabolism were stably maintained for 5 d in the hepatocytes cultured on fructose-chitosan. In sharp contrast, hepatocytes attached on a collagen-coated surface underwent a severe morphological change, from spherical to flat, and lost almost all their lidocaine-removal activity within 5d. A very thin fructose-chitosan layer was also applied onto the collagen-coated surfaces of polystyrene plates and a dextran microcarrier by crosslinking free amino groups in the chitosan and collagen with glutaraldehyde to fix the thin layer. Hepatocytes on the fructose-chitosan-coated surface retained their spherical shape, masking the cell-flattening effect of the collagen layer. Perfusion culture was then carried out using a hollow-fiber cartridge in which hepatocytes attached on fructose-chitosan-coated microcarriers were suspended in the extracapillary space: the liver-specific functions were stably maintained during 4d of the culture. A fructose-chitosan-coated surface thus appears to be a very promising scaffold for hepatocyte attachment which can be used in cellular biological studies of liver functions, especially in relation to cytochrome P450, as well as in bioartificial liver support systems.