0106 - HCT-8 [HRT-18]

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BCRJ Code 0106
Cell Line HCT-8 [HRT-18]
Species Homo sapiens
Vulgar Name Human
Tissue Colon
Morphology Epithelial
Disease Ileocecal Colorectal Adenocarcinoma
Growth Properties Adherent
Sex Male
Age/Ethinicity 67 Year /
Tumor Formation: Yes, in nude mice Tumors developed within 21 days at 100% frequency (5/5) in nude mice inoculated subcutaneously with 107 cells
Products carcinoembryonic antigen (CEA) 0.5 ng/10 exp6 cells/10 days; alkaline phosphatase; keratin
Biosafety 1
Addtional Info The HCT-8 line is identical to the HRT-18 cell line. The cells are positive for keratin by immunoperoxidase staining.
Culture Medium RPMI 1640 medium with 2 mM L-glutamine, 4.5 g/L glucose and 10% of fetal bovine serum.
Subculturing Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. T-75 flasks are recommended for subculturing this product. 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. 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 Every 2 to 3 days
Subculturing Subcultivation Ratio 1:4 to 1:8
Culture Conditions Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37°C
Cryopreservation 95% FBS + 5% DMSO (Dimethyl sulfoxide)
Thawing Frozen Cells SAFETY PRECAUTION: It is strongly recommended to always wear protective gloves, clothing, and a full-face mask when handling frozen vials. Some vials may leak when submerged in liquid nitrogen, allowing nitrogen to slowly enter the vial. Upon thawing, the conversion of liquid nitrogen back to its gas phase may cause the vial to explode or eject its cap with significant force, creating flying debris.
  1. Thaw the vial by gently agitating it in a 37°C water bath. To minimize contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
  2. Remove the vial from the water bath as soon as its contents are thawed and decontaminate it by dipping in or spraying with 70% ethanol. From this point, all operations must be performed under strict aseptic conditions.
  3. For cells sensitive to DMSO, it is recommended to remove the cryoprotective agent immediately. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge at approximately 125 × g for 5 to 7 minutes.
  4. Discard the supernatant and resuspend the cell pellet in the recommended complete medium (see specific batch information for the appropriate dilution ratio).
  5. Incubate the culture under appropriate atmospheric and temperature conditions (see "Culture Conditions" for this cell line).

NOTE: It is important to avoid excessive alkalinity of the medium during cell recovery. To minimize this risk, it is recommended to place the culture vessel containing the growth medium in the incubator for at least 15 minutes before adding the vial contents. This allows the medium to stabilize at its normal pH (7.0 to 7.6).
References Tompkins WA, et al. Cultural and antigenic properties of newly established cell strains derived from adenocarcinomas of the human colon and rectum. J. Natl. Cancer Inst. 52: 1101-1110, 1974. PubMed: 4826581 Nelson-Rees WA, et al. Distinctive banded marker chromosomes of human tumor cell lines. Int. J. Cancer 16: 74-82, 1975. PubMed: 1058173 White LJ, et al. Attachment and entry of recombinant norwalk virus capsids to cultured human and animal cell lines. J. Virol. 70: 6589-6597, 1996. PubMed: 8794293
Depositors Jose Paulo Leite, Instituto Oswaldo Cruz, Rio de Janeiro.
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