Cells, tumor trypsinized

For tumor cell lines, the cells were trypsinized, suspended and counted, then diluted to about 10 and plated onto Corning 1 ml well plates. The plates were incubated overnight in a lOOjt humidity carbon dioxide incubator and the next day the Dulbecco s Modified Eagle Medium (DMEM) is sucked off and substituted with DMEM containing various microgram quantities of... 

Tumor cells. EMT6 cells were grown as a monolayer culture in DMEM medium containing 20% fetal calf serum (27). Cells were detached from the plate by trypsin-EDTA treatment and washed in PBS. A total of 5 x 103 cells were injected per mouse via the tail vein of Balb/c mice (6-8 weeks old) to induce experimental lung metastatic tumors. Immunoliposomes were injected iv 2 and 4 days after the tumor cell injection. The survival of mice was followed over the next 60 days. 

To cite an example, this strategy was also used to activate methotrexate-Phe (6.36, R=Phe) and other methotrexate-a-peptides in the vicinity of tumor cells [64], Carboxypeptidase A is normally synthesized as a zymogen that is inactive without proteolytic removal of its propeptide end by trypsin. To adapt this system to GDEPT, a mutant form of the enzyme (CPASX3) was... 

Metastasis 2. Medium for tumor cell culture, e.g., Minimal Essential Medium Eagle (MEM) (Gibco, Invitrogen) 3. 10X Trypsin/EDTA dilute to final concentration lx with PBS (Sigma-Aldrich) 4. Standardized tumor cell suspension, radiolabeled 5. Hank s balanced salt solution, Ca++ and Mg++ free (CMF-HBSS) (Sigma-Aldrich, H-2387) 6. Trypan blue stain 7. Mouse vice 8. Heat lamps... 

Trypsinization in excess of 1 min has been reported to decrease lung colony formation and should therefore be avoided (13). In other studies it has been shown that comparing the incidence of metastasis from the injection of one predetermined dose of tumor cells does not allow an analysis of their relative metastatic capacities. Reproducible and meaningful results require studies that introduce increasing numbers of viable tumor cells admixed with a constant number ofnon-tumorigenic (X-irradiated) carrier cells (15) (see Note 4). 

Add CMF-HBSS to the flask to disperse cells by repeated pipetting over the monolayer surface. In addition, this dilutes the trypsin, reducing degradation of the tumor cell membrane. 

Prolonged and unnecessary enzymatic treatment, i.e., trypsinization of tumor cells, can also alter their survival and metastatic behavior in vivo. Moreover, viability tests (trypan blue exclusion) and even plating efficiency in vitro do not predict or correlate with the in vivo biological behavior of trypsinized cells. 

The Bowman-Birk inhibitor also blocks the transformation of C H/10T1/2 cells (18). This raises the speculation that BB may represent a direct acting nutritionally relevant anticarcinogen particularly in the case of colon cancer. In this regard it was recently reported that e-aminocaproic acid (a trypsin inhibitor) inhibits dimethylhydrazine-induced colon tumors in mice (22). 

Kobayashi H, Gotoh J, Hirashima Y, Terao T. Inter-alpha-trypsin inhibitor bound to tumor cells is cleaved into the heavy chains and the light chain on the cell surface. J Biol Chem 1996 271 11362-11367. 

Janssen U, Thomas G, Giant T, Phillips A. Expression of inter-alpha-trypsin inhibitor and tumor necrosis factor-stimulated gene 6 in renal proximal tubular epithelial cells. Kidney... 

Kobayashi H, Sugino D, Terao T. Urinary trypsin inhibitor, a Kunitz-type protease inhibitor, modulates tumor necrosis factor-stimulated activation and translocation of protein kinase C in U937 cells. Int J Oncol 1998 12 95-105. 

Shinohara H, Kobayashi H, Hirashima Y, Ohi H, Terao T. Urinary trypsin inhibitor (UTI) efficiently inhibits tumor cell invasion and metastasis in the experimental and spontaneous model. J Jpn Soc Cancer Ther 1996 3 186-195. 

Primary cells can be defined as cells taken directly from an organism and adapted to survive and eventually grow in culture, at least for a limited period. In fact, with the exception of some cells derived from tumors, primary cells have limited lifespans in culture. Cells can be isolated from tissues for ex vivo culture in several ways. They can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase that break down extracellular matrices. Alternatively, in the explant culture method, pieces of tissue are placed in growth media and the cells that grow are available for culture. 

Tumor cells are detached by EDTA treatment, rinsed with PBS and diluted in PBS at approximately 10 /ml. 100 /rl of the cell suspension are plated onto platelets and incubated for 1 h at 37 C. After washing three times with PBS, adherent tumor cells are removed by trypsin/EDTA treatment, and counted with an hemocytometer under phase microscopy. Alternatively, tumor cells can be radioac-... 

Aggregation assay. Tumor cells are detached from the culture dish by EDTA treatment (if trypsin has to be used, then a time for recovery in serum containing medium should be allowed before the aggregation assay) and diluted at 10 /ml in DMEM-2%FCS. 

EC monolayers obtained after growth on either collagen-coated glass coverslips, or tissue culture multiwell plates as previously described (Sections 2.3.3.1 and 2.3.3.2), are rinsed several times in serum Iree medium, and then incubated 1 h at 37°C with assay medium (usually serum free culture medium supplemented with 0.5-1% BSA), before they are used as substrates for the adhesion assay. Tumor cells are detached from culture dishes either by EDTA or by trypsin treatment. In the latter case, trypsinized cells should be allowed to recover from the enzyme treatment in their growth medium for 30 min at 37°C. After rinsing in serum-free medium, labeled tumor cells are resuspended at the desired concentration in the assay medium and plated onto the EC monolayer at a cell concentration ranging between 10 and 2 x 10 /cm. Incubation is... 

Cell and tissue implants Cell suspensions are obtained by trypsinization of confluent cell monolayers. Five microliters containing 2 X 10 cells in medium supplemented with 10% serum are introduced in the corneal micropocket. When the over expression of growth factors by stable transfection of a specific cDNA is studied, one eye is implanted with transfected cells and the other with the wild type cell line. A 0ien tissue samples are tested, samples of 2-3 mg are obtained by cutting the original fragments under sterile conditions. The angiogenic activity of tumor samples is compared with macroscopically healthy tissue. 

Ricin (castor bean) immunotoxin has been developed to attack the CD5 T-cell antigen (present in T-cell and some B-cell malignancies) as well as the interleukin-2 receptor of cancerous tumors. Ne-urobiological applications of ricin involve the study of brain function via lesioning. It is also used as a reagent for pepsin and trypsin, and as a commercial mole killer. Historically, it has been used as a biochemical warfare agent. 

Rat F98 glioma cells were from R. Goodman, Ohio State University (Columbus, OH). The 9L gliosarcoma line was obtained from the Brain Tumor Research Center, University of California, San Francisco. Cells were maintained in 10% fetal calf serum in DMEM supplemented with penicillin/streptomycin and tested by the Gen-Probe Rapid Detection System (Fisher Scientific) to rule out mycoplasma contamination. Cells were harvested with 0.25% trypsin, counted, and resuspended in DMEM solution before intracranial implantation. 

DAs are typically negative for pancreatic enzymes such as trypsin, chymotrypsin, and lipase °76 unless there is a mixed acinar component, which is uncommon. They also fail to label with endocrine markers however, in 30% of DAs there are scattered, possibly non-neoplastic, endocrine cells in close association with the neoplastic cells, which can be highlighted with immunostains for chromogranin A, synaptophysin, and NSE.2 2 77-79 latter two markers can occasionally show more diffuse expression, which should not be regarded as evidence of neuroendocrine differentiation if the tumor is an otherwise conventional adenocarcinoma. 

The tumors may arise sporadically or in patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome.Current experience is too limited to determine their biologic behavior and prognosis however, in one study, the patients were found to have an improved survival rate relative to those with DAs. ° Immunohistochemically, the epithelioid cells are labeled by antibodies to cytokeratins whereas trypsin, chymotrypsin, lipase, chromogranin, and synaptophysin are usually negative. CD3 antibody highlights the presence of numerous intratumoral T lymphocytes. Rare examples also contain Epstein-Barr virus RNA. ... 

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