The potential use of porcine hepatocytes in a bioartificial liver device requires large quantities of viable and highly active cells. To facilitate the scaling up of the system, liver specific activities of hepatocytes should be maximized. One way of enhancing the specific activities is to cultivate hepatocytes as multicellular spheroids. Freshly isolated porcine hepatocytes form spheroids when cultivated in suspended cultures. These spheroids exhibit higher activities for a number of liver specific functions compared to hepatocytes cultivated as monolayers. However, these activities decreased in a few days in culture. Entrappment of spheroids in collagen gel sustained their metabolic activities at a stable level over 21 days. Production of albumin and urea by spheroid hepatocytes entrapped in collagen gels were 2 to 3 times higher than those by freshly isolated single cells. P-450 activity was demonstrated by metabolism of lidocaine to its main metabolite, monoethylglycinexylidide. Phase II drug metabolism was demonstrated by glucuronidation of 4-methylumbelliferone. This work shows that porcine hepatocyte spheroids entrapped in collagen maintain differentiated functions for an extended time period. Such hepatocyte spheroid entrappment system may facilitate the development of a bioartificial liver support device.
Production of recombinant human acetylcholinesterase (AChE) by a high producer human embryonic kidney cell line (293) was evaluated by three main cell propagation systems; surface propagator, fixed-bed reactor and stirred microcarrier cultures. The recombinant cell line expresses AChE levels as high as 10-20 mg/l/day. System productivities in either the surface propagator (multitray system), or in the fixed-bed reactor (polyurethane macroporous sponges) were 4-8 mg AChE/l/day during a production period of 8 days. Similar productive rates, yet longer production periods (up to 22 days), were obtained in microcarrier (MC) cultures using either polystyrene beads (Biosilon); collagen-coated dextran beads (Cytodex-3); or gelatin macroporous beads (Cultispher-G). Best results were obtained in an aggregate culture using cellulose beads charged with diethylaminoethyl (DEAE) groups, (Servacel), as carriers. In this culture, a system productivity of 6-10 mg/l/day was maintained for 28 days.
The high incidence of viral infections in patients with lymphocytic leukemia is well documented, but the role played by interferon in the pathogenesis of such infections is not known. In this study, we investigated the possibility that gamma (gamma) interferon production, induced by phytohemagglutinin (PHA) might be impaired in leukocytes from patients with acute lymphocytic leukemia (ALL). We also compared this response with alpha (alpha) interferon production, and with PHA-stimulated lymphocyte transformation. We have shown that the gamma interferon response of leukocytes from patients, both in relapse and in remission, was markedly lower than in leukocytes from normal donors. However, the alpha interferon response in leukocytes from the patients was normal. In contrast the defective gamma interferon response to PHA stimulation of cells from patients in remission, lymphocyte transformation by PHA was normal. Lymphocytes from patients in relapse has a delayed response. Our findings suggest (1) that the defective gamma interferon response which occurs in cells from patients with ALL, both in relapse and remission, contributes to increased susceptibility to viral infections, (2) that alpha interferon may not be the optimal type of interferon for treatment of certain viral infections, and (3) that different triggering mechanisms, or different receptors, exist for PHA-induced gamma interferon production and for lymphocyte transformation in cells of patients with ALL.
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