No abstract

Fetal liver epithelial cells (FLEC) are valuable for liver cell therapy and tissue engineering, but methods for culture and characterization of these cells are not well developed. This work explores the influence of multiple soluble factors on FLEC, with the long-term goal of developing an optimal culture system to generate functional liver tissue. Our comparative analysis suggests hepatocyte growth factor (HGF) is required throughout the culture period. In the presence of HGF, addition of oncostatin M (OSM) at culture initiation results in concurrent growth and maturation, while constant presence of protective agents like ascorbic acid enhances cell survival. Study observations led to the development of a culture medium that provided optimal growth and hepatic differentiation conditions. FLEC expansion was observed to be ~2 fold of that under standard conditions, albumin secretion rate was 2 – 3 times greater than maximal values obtained with other media, and the highest level of glycogen accumulation among all conditions was observed with the developed medium. Our findings serve to advance culture methods for liver progenitors in cell therapy and tissue engineering applications.

No abstract

CD8+ cytotoxic T‐lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide‐specific CD8+ T‐lymphocytes from the peripheral blood of healthy, human trophic leukemia virus‐1 (HTLV‐1) seronegative human leucocyte antigen (HLA)‐A*0201 individuals. A combination of density‐based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen‐presenting cells (APCs), and T‐lymphocytes. The DCs were pulsed with HLA‐A*0201 binding peptides and cultured with autologous T‐lymphocytes to generate peptide‐specific CTLs. The CTLs were generated against a nine‐amino‐acid peptide from the Tax protein of HTLV‐1. The CTLs were expanded according to a restimulation schedule employing peptide‐pulsed autologous monocytes and low‐dose interleukin‐2 (IL‐2) to numbers in excess of 100 × 106 cells following 5 weeks of culture. Expanded cells contained primarily CD3+ T‐cells, of which CD8+ T‐lymphocytes constituted greater than two‐thirds of the cell population. Obtained CTLs exhibited potent antigen‐specific lysis of peptide‐pulsed target cells in a dose‐dependent fashion in in vitro 51Cr release cytotoxicity assay. This antigen‐specific killing was shown to be HLA class I restricted and mediated by CD8+ T‐lymphocytes. Since the T‐lymphocytes were obtained from HTLV‐1 seronegative donors, the generation of peptide‐specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. © 1996 John Wiley & Sons, Inc.