||Stomach; Derived From Metastatic Site: Liver
||yes, the cells form tumors in athymic nude mice. [PubMed: 2158397]
||NCI-N87 cells express the surface glycoproteins carcinoembryonic antigen (CEA) and TAG 72, and are L-dopa decarboxylase (DDC) negative. They were minimally positive for vasoactive intestinal peptide (VIP) receptors and lacked gastrin receptors. They were found to express receptors for muscarinic cholinergic agents. No evidence of amplification or rearrangements was noted with the N-myc, L-myc, myb and EGF receptor genes. The cell line expressed levels of c-myc and c-erb-B 2 RNA that were comparable to other cell lines.There was no expression of the following genes: N-myc, L-myc, c-cis, IGF-2, or gastrin releasing peptide. NCI-N87 cells have a reported plating efficiency of 4.3%.
||RPMI-1640 medium modified to contain 2 mM L-glutamine, 4500 mg/L glucose with fetal bovine serum to a final concentration of 10%.
||Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
Remove and discard culture medium.
Briefly rinse the cell layer with PBS without calcium and magnesium to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C.
Population Doubling Time: 47 hrs
NOTE: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 12 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 6th edition, published by Alan R. Liss, N.Y., 2010.
|Subculturing Medium Renewal
||Two to three times weekly
|Subculturing Subcultivation Ratio
||1:3 to 1:4 is recommended
||Atmosphere: air, 95%; carbon dioxide (CO2), 5%
||95% FBS + 5% DMSO (Dimethyl sulfoxide)
|Thawing Frozen Cells
||SAFETY PRECAUTION: Is highly recommend that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that
some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris.
1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the Oring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. For cells that are sensitive to DMSO is recommended that the cryoprotective agent be removed immediately. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 7 minutes.
4.Discard the supernatant and Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio).
5. Incubate the culture in a appropriate atmosphere and temperature (see "Culture Conditions" for this cell line).
NOTE: It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
||Park JG, et al. Characteristics of cell lines established from human gastric carcinoma. Cancer Res. 50: 2773-2780, 1990. PubMed: 2158397
NCI-Navy Medical Oncology Branch Cell Line Supplement. J. Cell. Biochem. suppl. 24: 1996.
NCI-N87 is a gastric carcinoma cell line derived in 1976 by A. Gazdar and associates at the National Cancer Institute from a liver metastasis of a well differentiated carcinoma of the stomach taken prior to cytotoxic therapy. The tumor was passaged as a xenograft in athymic nude mice for three passages before the cell line was established.
||Lidia Moreira Lima - Universidade Federal Do Rio De Janeiro