Liver tissue fragments obtained from males are the most promising source of human hepatocytes for cell-based therapies - Flow cytometric analysis of albumin expression.

Cell-based therapies that could provide an alternative treatment for the end-stage liver disease require an adequate source of functional hepatocytes. There is little scientific evidence for the influence of patient's age, sex, and chemotherapy on the cell isolation efficiency and metabolic activity of the harvested hepatocytes. The purpose of this study was to investigate whether hepatocytes derived from different sources display differential viability and biosynthetic capacity. Liver cells were isolated from 41 different human tissue specimens. Hepatocytes were labeled using specific antibodies and analyzed using flow cytometry. Multiparametric analysis of the acquired data revealed statistically significant differences between some studied groups of patients. Generally, populations of cells isolated from the male specimens had greater percentage of biosynthetically active hepatocytes than those from the female ones regardless of age and previous chemotherapy of the patient. Based on the albumin staining (and partially on the α-1-antitrypsin labeling) after donor liver exclusion (6 out of 41 samples), our results indicated that: 1. samples obtained from males gave a greater percentage of active hepatocytes than those from females (p = 0.034), and 2. specimens from the males after chemotherapy greater than those from the treated females (p = 0.032).

Dried human skin fibroblasts as a new substratum for functional culture of hepatic cells.

The primary hepatocytes culture is still one of the main challenges in toxicology studies in the drug discovery process, development of in vitro models to study liver function, and cell-based therapies. Isolated hepatocytes display a rapid decline in viability and liver-specific functions including albumin production, conversion of ammonia to urea, and activity of the drug metabolizing enzymes. A number of methods have been developed in order to maintain hepatocytes in their highly differentiated state in vitro. Optimization of culture conditions includes a variety of media formulations and supplements, growth surface coating with the components of extracellular matrix or with synthetic polymers, three-dimensional growth scaffolds and decellularized tissues, and coculture with other cell types required for the normal cell-cell interactions. Here we propose a new substratum for hepatic cells made by drying confluent human skin fibroblasts' culture. This growth surface coating, prepared using maximally simplified procedure, combines the advantages of the use of extracellular matrices and growth factors/cytokines secreted by the feeder layer cells. In comparison to the hepatoma cells grown on a regular tissue culture plastic, cells cultured on the dried fibroblasts were able to synthesize albumin in larger quantities and to form greater number of apical vacuoles. Unlike the coculture with the living feeder layer cells, the number of cells grown on the new substratum was not reduced after fourteen days of culture. This fact could make the dried fibroblasts coating an ideal candidate for the substrate for non-dividing human hepatocytes.

Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines.

Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests.

Evaluation of the effects of antibiotics on cytotoxicity of EGFP and DsRed2 fluorescent proteins used for stable cell labeling.

The use of fluorescent markers has proven to be an attractive tool in biological imaging. However, its usefulness may be confined by the cytotoxicity of the fluorescent proteins. In this article, for the first time, we have examined an influence of the antibiotics present in culture medium on cytotoxicity of the EGFP and DsRed2 markers used for whole-cell labeling. Results showed that doxycycline negatively affected albumin synthesis in DsRed2-expressing hepatoma cells, and that both hepatoma cells and human skin fibroblasts, labeled with this protein, were characterized by the lowered growth rates. Thus, the cytotoxic effect of fluorescent markers depends on both protein used for cell labeling and on growth conditions that may cause cell stress.

Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models.

Hepatic/nonhepatic cell cocultures are widely used in studies on the role of homo- and heterotypic interactions in liver physiology and pathophysiology. In this article, for the first time, establishment of the coculture model employing hepatoma C3A cells and human skin fibroblasts, stably expressing fluorescent markers, is described. Suitability of the model in studying coculture conditions using fluorescence microscopy and flow cytometry was examined. C3A cells spontaneously formed island-like growth patterns surrounded by fibroblasts. The "islands" size and resulting intensity of the homo- and heterotypic interactions can easily be tuned by applying various plated cells ratios. We examined the capability of the hepatoma cells to produce albumin in hepatic/nonhepatic cell cocultures. The enzyme-linked immunosorbent assay (ELISA) tests showed that greater number of fibroblasts in coculture, resulting in smaller sizes of hepatoma "islands," and thus, a larger heterotypic interface, promoted higher albumin synthesis. The use of fluorescently labeled cells in flow cytometry measurements enabled us to separately gate two cell populations and to evaluate protein expression only in/on cells of interest. Flow cytometry confirmed ELISA results indicating the highest albumin production in hepatoma cells cocultured with the greatest number of fibroblasts and the inhibited protein synthesis in coculture with osteosarcoma cells.