Ward D. Peterson

Performance of plasma-perfused, microencapsulated hepatocytes: Prospects for extracorporeal liver support

The growing success of liver transplantation and the shortage of donor livers has turned attention to the possibility of utilizing hepatocytes within artificial liver support systems to allow time for donor livers to become available and to improve the condition of patients with hepatic failure. This study evaluated encapsulated hepatocytes, a technology which might allow the possibility of using xenogenic or human hepatoma cells. Rabbit hepatocytes were encapsulated using the ionic polysaccharides carboxymethylcellulose, chondroitin sulfate A, chitosan, and polygalacturonic acid. Encapsulated cells were maintained in perfusion culture for at least 6 days in heparinized, normal human plasma or in a defined culture medium. Parallel cultures of plated hepatocytes were also conducted. The metabolic capability of the cells was evaluated by following the rates of urea, albumin, and transferrin synthesis and the transformation rate of the drug antipyrine. Protein synthesis and ureogenesis in plasma were depressed from the levels expressed in defined culture medium. Drug detoxification as measured by antipyrine metabolism appeared to be enhanced in plasma. We conclude that encapsulated rabbit hepatocytes retain significant levels of function for at least 6 days of perfusion with human plasma, suggesting the feasibility of this technology as a potential method of short-term liver support.

Cell Characterization by Use of Multiple Genetic Markers

The extensive use of cell cultures for diverse research purposes is one of the truly great international growth industries. With the proliferation of cells comes a responsibility for monitoring them for inter- and intraspecies characteristics. We use multiple genetic markers for cell identification, i.e. species specific antigens, isozymic phenotypes, chromosomal complement, and HL-A haplotypes. The methodologies employed are briefly described, and various examples cited to show how these markers can be utilized for cell line monitoring. Data are summarized from 275 cultures sent to our laboratory for analysis during the past eighteen months. The data show that, overall, 35% of the cultures received were contaminated. The majority of cell cultures submitted were human cell lines. We found that 36% of these cultures were cross contaminated; 25% by cells of another species and 11% by another human cell line. This high incidence of inter- and intraspecies contamination underscores the importance of frequent monitoring of cell cultures.

Long-term proliferation of mouse thymic epithelial cells in culture

SummaryEpithelial cells from the normal mouse thymus were successfully cultivated on tissue culture plastic when plated with lethally irradiated support cells of the LA7 rat mammary tumor line. As the irradiated LA7 cells slowly decreased in number the thymus cells proliferated concomitantly to form a confluent monolayer. The cells now in culture have been subcultured 8 times, have doubled in number at least 30 times, and are still proliferating vigorously. The culture technique also supported clonal growth from a single cell, and nine clones have been isolated. The colony-forming efficiency of thymic cells plated at low concentrations was about 8%. These cultures were never overgrown by fibroblasts. The thymus cells were characterized as epithelial by the presence of cytoplasmic keratin and numerous desmosomes and tonofilaments. They were shown to be mouse cells by immunocytochemistry with species specific antibodies, by isoenzyme analysis, and by karyology. The cells stained when reacted with antibodies to tubulin, vimentin, and actin, but not with antibodies to Thy-1.2, Lyt-1, Lyt-2, Ia, or H-2 proteins. More than 85% of the cells had a normal mouse diploid chromosome number of 40. This culture technique opens the way for future studies of T-cell education with homogeneous thymic epithelial cell populations both in vitro and after reimplantation into genetically defined strains of mice.

Epstein-Barr Virus Infection of Cryopreserved Umbilical Cord Blood Lymphocytes

Summary We have described the application of cryopreservation technology to freeze storage of human primary umbilical cord blood lymphocytes and have shown that the cryopreserved cells remain as sensitive to EB virus infection and transformation as the fresh autologous lymphocytes. The methodology has been satisfactorily applied to primary peripheral blood lymphocytes from adult human sources, and has been a convenient time and resource saving advance.

Development of a new human breast cancer cell line Ia-270.

SummaryA new human breast cancer cell line (Ia-270) has been isolated from a malignant pleural effusion from a woman with metastatic infiltrating ductal carcinoma of the breast. This cell line contains cytoplasmic estrogen (ER) and progesterone (PR) receptors. Following estradiol (E2) administration, PR synthesis is augmented and a higher level of saturation density is reached. In an athymic mouse, the cell line produced a tumor morphologically similar to the primary tumor. The results of isoenzyme and karyotype analyses demonstrate Ia-270 to be of human origin and free of HeLa cell contamination. The cell line has been maintained in continuous culture since April 1982 and may provide a usefulin vitro system for studying the biology of human breast cancer.

EPSTEIN-BARR VIRUS |[lpar]|EBV|[rpar]| NEGATIVE HUMAN MALIGNANT T LYMPHOCYTE CELL LINES

Most established human lymphocyte cell lines have properties of B cells and are EBV positive. Recently a few lymphoblastoid lines have been shown to have T cell properties (Minowada, J., et al, J.N.C.I. 49:891, 1972) (J. Kaplan, et al, Cancer Res. in press, 1974.) This report describes studies of the malignant lymphoblast cell lines CCRF-CEM and HSB-2, derived from two children with leukemia secondary to lymphosarcoma. Like normal T cells these two lines form more rosettes with sheep erythrocytes at 4° than at 37°. Rosette formation is inhibited to a greater degree by rabbit antiserum to T cell lines than by antiserum to B cell lines. The cells lack complement receptors and surface immunoglobulins. When examined for EBV capsid antigen and EBV nuclear antigen, an indicator of EBV genome, CCRF-CEM and HSB-2 are negative, whereas all B cell lines so studied are positive for EBV nuclear antigen.These findings indicate that lines CCRF-CEM and HSB-2 are T cells and that they are EBV negative. Perhaps they possess genetic material of some other virus which, like EBV, induces both sustained in vitro cell growth and in vivo tumors. If so, the availability of these malignant T lymphocyte lines may facilitate the search for a human leukemia-lymphocyte virus.