Nonperfused attachment systems for cell cultivation.

The pressing need for large-scale culture methods has prompted efforts to develop vessels which will accommodate the growth of large numbers of anchorage-dependent cells. The large-scale "cell factories" have been used to grow viruses for vaccine production, nucleic acid studies, and various cancer research projects. Two particular types of culture vessels used for large-scale production of anchorage-dependent cells were discussed and examples of their use in vaccine production given. Development of these large-scale culture systems has enabled the pharmaceutical companies to (1) meet the ever-increasing demands worldwide for vaccine, (2) employ a production process that produces a cost-efficient vaccine product in cell culture, and (3) produce large volumes of bulk vaccine at a single campaign, thus allowing multiple usage of a single production unit over the course of a year. The type of large-scale culture vessel used depends on the purpose of the culture and the type of cells that are to be grown in the vessel. The unit process vessel, with its multidisks, provides superior surface area, but because of the stainless steel housing, the cells cannot be monitored microscopically. Additionally, the unit process vessel can be equipped with a jacket for precise temperature regulation and thus eliminate the need for incubators. The roller bottles, on the other hand, allow microscopic monitoring of the growing cells but are limited in surface area available for growth, and the volume of harvest fluid obtainable from a bottle is very limited. It should be noted that handling of roller bottles is labor-intensive and requires many more manipulations and hence more personnel than employing unit process-type vessels for vaccine-manufacturing operations. The use of mass cultivation techniques in cancer research has also been of immense benefit. The production of large numbers of neoplastic cells has provided researchers with the raw materials to perform molecular biology experiments such as DNA sequencing, which required milligram quantities of cells before sufficient quantities of DNA could be isolated to allow sequencing studies. Additionally, the technique of mass cultivation also provided sufficient quantities of cells for inoculation into either nude mice or immunosuppressed hamsters in an attempt to determine the tumor induction capacity of the cultured cells. Additional characterization studies which require large numbers of cell, enzyme, and isoenzyme profiles can also be performed on cells grown in roller bottle cultures. There are numerous factors which limit the productivity of mass cultivation systems and the scientist should be aware of their existence.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholesterol accumulation in heterotransplanted renal cell cancer.

To date there have been no in vivo studies of the known propensity of human renal cell cancer (RCC) to accumulate large amounts of cholesterol. Caki-1, a human RCC line, was grown in the cheek pouches of immunosuppressed golden Syrian hamsters. Analysis of cholesterol levels revealed 2 to 4 times as much cholesterol in the heterotransplants as in any of the host tissues. After intraperitoneal injection of tumor-bearing hamsters with 4-14C-cholesterol, the tumors accumulated less radioactivity than did hamster liver and no more radioactivity than did hamster kidney or cheek pouch. It appears that accumulation of cholesterol by human RCC is not dependent on extensive uptake of extracellular cholesterol. Instead, an intrinsic defect in the cellular regulation of cholesterol synthesis and efflux is more likely to be responsible.

Carcinoembryonic antigen-like substance derived from human prostate.

Carcinoembryonic antigen-like substance, previously detected in large amounts in the medium from cultures of human prostatic epithelial cells, also is present in extracts of benign and malignant human prostate. By column chromatography, the prostate-derived carcinoembryonic antigen-like substance derived from cultured prostate is the same as that in tissue extracts and is distinctly different from colon-derived carcinoembryonic antigen. The molecular weight of prostate-derived carcinoembryonic antigen-like substance is estimated to be greater than 5 x 10(5). Prostate-derived carcinoembryonic antigen-like substance may be a prostate-specific substance.

Transitional cell cancer: establishment and characterization of cell lines.

Eleven long-term (in culture more than 1 yr) cell lines were established from surgical specimens of human TCC. Characterization studies performed on the individual cell lines showed that each 1) demonstrated an abnormal human karyotype, 2) grew in soft agar, 3) exhibited rapid growth and multilayering 4) was free from microbial and HeLa cell contamination, 5) produced tumors in cheek pouches of immunosuppressed Syrian golden hamsters, 6) contained ultrastructural features consistently found in epithelial cells in culture, and 7) could be grown to high cell densities in roller-bottle cultures.

Transitional cell cancer: fluorescent antibody binding to tumor cells in vitro.

The IFA assay has been used to detect antigen--antibody reactions between sera from patients with TCC and TCC cell lines established in our laboratory. A positive immunofluorescence reaction, consisting of perinuclear staining, was seen in 86% of the sera from TCC patients exposed to 253J cells. A positive reaction was detection in only 8% of the sera from healthy donors, in 19% of that from patients with nonmalignant urinary tract disease, and in 31% of the sera from patients with urinary tract tumors other than TCC when exposed to 253J cells. No positive reaction was detected when these same sera were exposed to either WI-38 or normal human kidney cells. The positive fluorescence could be removed from TCC sera by absorption with TCC surgical specimens but not by absorption with nontumor surgical specimens.

In vitro cultivation of human renal cell cancer. II. Characterization of cell lines.

Two cell lines derived from primary human renal-cell cancers (RCC) have been established and characterized. Cell line 786-O has been in culture for longer than 1 year and has been subcultured more than 50 times. It has a doubling time of 45 hr and a hypertriploid karyotype and possesses a Y chromosome. Cell line 769-P also has been in culture for longer than 1 year. It has been subcultured 50 times and has a doubling time of 35 hr and a hypodiploid karyotype. Cells from both lines are epithelial, and they produce tumors in the cheek pouches of immunosuppressed hamsters. Neither cell line is contaminated with Mycoplasma. Cells of the two lines can be distinguished from HeLa cells both by their karyotypes and by the mobility patterns of their isoenzymes of glucose-6-phosphate dehydrogenase.

Biochemical markers of cultured human prostatic epithelium.

Medium from primary cultures of human prostatic cells was examined for polyamines and carcinoembryonic antigen. Spermine was elevated in medium from 5 of 17 epithelial cell cultures but was not found to be a specific marker for cultured human prostatic epithelial cells. Carcinoembryonic antigen was elevated in all 27 prostate epithelial-cell culture fluids tested, 24 of which had more than 20 ng. carcinoembryonic antigen per milliliter. Medium from other cultured human cells (prostate fibroblasts, genitourinary tumor and non-tumor cells), as well as control medium, contained less than 1 ng. carcinoembryonic antigen per milliliter. Carcinoembryonic antigen, as measured in this study, is a way to demonstrate the presence of human prostatic epithelial cells in culture.

Investigation of human urogenital tract tumors of papovavirus etiology: brief communication.

Cells cultured from human urogenital cancer and other cancers as well as cells from noncancerous tissues were examined by immunofluorescent staining with antibodies to T-antigens and capsid antigens of papovaviruses BK virus (BKV), JC virus, and simian virus 40(SV40), and to capsid antigens of herpes simplex virus types 1 and 2 and human cytomegalovirus (CMV). Cells from early passage cultures of 123 primary tissues and from 14 continuous lines derived from transitional or renal cell carcinoma were tested. None of the cell preparations was specifically stained with any of the antisera. A serologic comparison of patients with bladder cancer, patients with prostate cancer, and normal control groups of BKV hemagglutination-inhibiting and SV40-neutralizing antibodies showed no differences among the 3 groups. None of the sera in the 3 groups had SV40 or BKV T-antibodies. In tests of supernatants of 35 primary cultures for presence of virus, a single isolation, that of a cytomegalovirus, was made. The study revealed no evidence that infection with papovaviruses of the SV40-polyoma subgroup has any part in the production of bladder and prostate cancer.

Properties of cell lines established from transitional cell cancers of the human urinary tract.

Characterization studies have been carried out on eight cell lines (253J, 192B, 639V, 647V, 486P, 575A,743E, and 751G) established from transitional cell cancers of the human urinary tract. Although subtle morphological differences exist among individual lines, each has an epithelial morphology and exhibits multilayering. The doubling times for the cells range from 20 to 56 hr, and at least a 1-to-3 split can be achieved when they are subcultured every 4th day. Karyotypic analysis revealed a hyperdiploid stemline for each cell line, and presence of a Y chromosome was confirmed by Q banding in five of the lines. The tumorigenic nature of the cell lines was demonstrated by their production of tumors in hamsters and confirmed by colony formation in agar. The transitional cell cancer lines were shown to be free of Mycoplasma, and their glucose-6-phosphate dehydrogenase mobility patterns and their Karyotypes prove that they are not HeLa cells.

Production of carcinoembryonic antigen by human prostate epithelial cells in vitro.

The medium from all 16 human prostate epithelial cell cultures tested contained elevated levels of carcinoembryonic antigen (CEA) or CEA-like substance; 11 of 16 had greater than 20 ng CEA/ml. In contrast, medium from cultures of other human tissues (prostate fibroblasts, genitourinary tumors, melanoma, and nontumor tissue), as well as media controls, contained less than 1 ng CEA/ml. Results indicated that CEA determination may provide a way to identify human prostatic epithelium in culture.

A comparison of four methods used to concentrate Rous sarcoma virus from tissue culture fluids.

Three methods of pelleting, pelleting followed by Pronase treatment, polyethylene glycol (PEG)-Pronase, and diaflo ultrafiltration (diafiltration) were used to concentrate RSV(RAV-1) from tissue culture fluids. Sucrose-gradient fractions containing virus preparations which had been concentrated by diafiltration or pelleting were heavily contaminated with amorphous debris. This debris was not present in similar, gradient-purified preparations that had been concentrated by the PEG-Pronase or pellet-Pronase methods. Maximum recovery of radiolabelled virus particles and virion-associated RNA-dependent DNA polymerase activity was obtained in gradient fractions containing virus concentrates prepared by the pellet-Pronase and PEG-Pronase methods. Although there were slight differences in recovery by these two methods, the advantages of the PEG-Pronase method make it the preferred method, especially when large volumes of tissue culture fluids are used.

In vitro cultivation of human renal cell cancer. I. Establishment of cells in culture.

A technique for initiating and propagating epithelial cell cultures of human renal cell cancer and adjacent nontumor kidney is described. Seventy-five percent of the tumors and 79% of the adjacent kidney specimens cultured with this method have shown initial outgrowth and have been subcultured at least once. Two renal cell cancer cultures initiated by this method have now been in continuous culture more than 6 months, have been subcultured 27 and 18 times, and now appear to be stable lines. The ability to establish long-term in vitro cultures of human renal cell cancers will facilitate studies concerning the immunoreactivity, cholesterol metabolism, the isolation of renal-cell-cancer-specific antigens, and in vitro chemotherapy testing and will further our understanding of the basic biology of human renal cell cancer.

Changes in cell-mediated cytotoxicity during the clinical course of patients with bladder carcinoma.

The conventional cell-mediated cytotoxicity assay was modified so that more useful estimates of cell-mediated immunity to transitional cell carcimoma of the urinary tract could be obtained. With this modified assay changes in the levels of cell-mediated immunity in transitional cell carcinoma patients to transitional cell carcinoma target cells were measured during the clinical course. Initial results from a small number of patients with invasive and non-invasive tumors showed that anti-transitional cell carcinoma cell-mediated immunity increased after the tumor was removed and during administration of Bacillus Calmette-Guerin.

In vitro cultivation of epithelial cells derived from tumors of the human urinary tract.

Finely minced explants from 54 TCC2 of the human urinary tract were cultured in vitro in an attempt to establish cell lines. Cells with epithelial morphology grew out from 48 tumor explants, and long-term cell cultures were established from 10. Six of the cell cultures have been maintained for over 18 months with 50 to 70 transfers and, therefore, are considered permanent cell lines. The epithelial cells in the established cultures are small, exhibit rapid doubling time, and show multilayering. The cells were examined both microscopically and by cultivation techniques, and they were found to be free from contaminating microorganisms, including Mycoplasma. The established cultures grow rapidly in roller bottles and, therefore, can be produced in large quantities. These cells also remain viable after being stored for 3 years in liquid nitrogen.

Concentration of Rous sarcoma virus from tissue culture fluids with polyethylene glycol.

Concentration of Rous sarcoma virus from tissue culture fluids with polyethylene glycol, with and without NaCl or dextran sulfate, resulted in significant and highly variable losses caused by entrapment of virus particles in proteinaceous debris. Treatment of concentrated preparations with Pronase greatly enhanced the recovery of virions. Maximum recovery of virus particles was obtained by the addition of 8% polyethylene glycol and 0.4 M NaCl to tissue culture fluids, followed by Pronase treatment of the concentrated virus preparations.

Technique for cultivation of transitional epithelium from mammalian urinary bladder.

A technique for initiating cultures of epithelial (urothelial) cells from mammalian urinary bladder has been described. Urothelial cells obtained by this method have been used to support replication of viruses and as controls for immunological, biochemical, chromosome, and electron microscopy studies. Both light and electron microscopic studies of cultured cells suggest that they are epithelial and not a mixed culture.