The influence of membrane molecular weight cutoff on a novel bioartificial liver.

Given the xenogeneic immune reaction relevant to the molecular weight cutoff of the membrane of a bioartificial liver (BAL) system, we investigated the influence of membrane molecular weight cutoff in our BAL system in this study. Acute liver failure in beagles was induced by d-galactosamine administration. Eight beagles were divided into two groups by the membrane molecular weight cutoff of the plasma component separator. Group 1 beagles were treated with BAL containing 200 kDa retention rating membrane. Group 2 beagles were treated with BAL containing 1200 kDa retention rating membrane. Each group underwent two 6-h BAL treatments that were performed on day 1 and day 21. The hemodynamic and hematologic response, humoral immune responses, and cytotoxic immune response to BAL therapy were studied before and after treatments. All beagles remained hemodynamically and hematologically stable during BAL treatments. BAL treatment was associated with a significant decline in levels of complement; however, a longer time of level maintenance was observed in Group 2. Group 2 beagles experienced a significant increase in levels of IgG and IgM after two BAL treatments. Significant levels of canine proteins were detected in BAL medium from Group 2; only trace levels of canine proteins were detected in BAL medium from Group 1. The posttreatment viability of co-culture cells in Group 2 was lower compared with Group 1, and the viability of co-culture cells after treatments was associated with deposition of canine proteins on the cells. Xenogeneic immune response was influenced by membrane molecular weight cutoff in the BAL.

Effects of membrane molecular weight cutoff on performance of a novel bioartificial liver.

Immunoisolation using semipermeable membranes has been incorporated into bioartificial liver (BAL) devices to separate cellular components of the recipient's immune system from the cells within the BAL device. This study was designed to explore the influence of membrane molecular weight cutoff on performance of the multilayer radial-flow BAL using porcine hepatocytes cocultured with mesenchymal stem cells. In this study, healthy beagles underwent 6-h treatment with a BAL containing membrane with 200 kDa retention rating or 1200 kDa retention rating. Functional markers of BAL performance were monitored before and after treatment, as well as cytotoxic immune response to BAL therapy. The results showed that hepatocyte performance levels such as albumin secretion, urea synthesis, and viability were all significantly higher in 200 kDa retention rating group compared with the 1200 kDa retention rating group after treatment (P < 0.05). Significant levels of canine proteins were detected in BAL medium from the 1200 kDa retention rating group. Fluorescence microscopy further verified that heavy deposition of canine IgG, IgM, and complement (C3) on coculture cells was obtained after BAL treatment in the 1200 kDa retention rating group. However, only trace deposits of canine immunoproteins were observed on coculture cells obtained from BAL in the 200 kDa retention rating group. Small membrane molecular weight cutoff of the BAL could reduce the transfer of xenoreactive antibodies into the BAL medium and improve the performance of the BAL.

Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-I collagen-coated poly(L-lactic acid).

Primary hepatocytes cultured in three dimensional tissue constructs composed of multicellular aggregates maintain normal differentiated cellular function in vitro while cultured monolayers do not. Here, we report a technique to induce hepatocyte aggregate formation using type-I collagen-coated poly(L-lactic acid) (PLLA) discrete aligned nanofibers (disAFs) by providing limited cell-substrate adhesion strength and restricting cell migration to uniaxial movement. Kinetics of aggregate formation, morphology and biochemical activities of rat hepatocyte aggregates were tested over a 15 day culture period. Evidence was provided that physical cues from disAFs quickly induced the formation of aggregates. After 3 days in culture, 88.3% of free hepatocytes on disAFs were incorporated into aggregates with an average diameter of 61 +/- 18 microm. Hepatocyte aggregates formed on disAFs displayed excellent cell retention, cell activity and stable functional expression in terms of albumin secretion, urea synthesis and phase I and II (CYP1A and UGT) metabolic enzyme activity compared to monolayer culture of hepatocytes on tissue culture plastic (TCP) with type-I collagen as well as on meshes of type-I collagen-coated PLLA random nanofibers (meshRFs). These results suggest that disAFs may be a suitable method to maintain large-scale hepatic cultures with high activity for tissue engineering research and potential therapeutic applications, such as bioartificial liver devices.

Electrospun chitosan nanofibers for hepatocyte culture.

In this study, we developed a method to obtain high surface area nanofiber meshes composed of chitosan of a number of molecular weights. This method required decreasing the viscosity and surface tension of the chitosan solution as well as optimization of the electrospinning parameters such as applied voltage and environmental humidity. These chitosan nanofiber meshes were developed as a culture substrate for hepatocytes. The fibers exhibited a uniform diameter distribution (average diameter: 112 nm) and FTIR results indicate that the chemical structure of chitosan is stable during the electrospinning process. The attachment, morphology and activity of hepatocytes cultured on the chitosan nanofiber meshes were tested. The results showed that the chitosan nanofibers are biocompatible with hepatocytes and that these chitosan nanofiber meshes could be useful tissue culture substrates for various applications, including bioartificial liver-assist devices and tissue engineering for liver regeneration.