| Cell Line | Human keratinocyte (hKT) |
| BBRJ Code | nh-skp-KT0009 |
| Product Type | Primary cells |
| Species | Homo sapiens |
| Vulgar Name | Human |
| Tissue | Skin, Foreskin |
| Cell Type | Keratinocyte |
| Morphology | Polygonal |
| Growth Properties | Adherent |
| Sex | Male |
| Age/Ethinicity | 7 Year / Brown |
| Derivation | Established from human foreskin |
| Applications | In vitro Assays for Research and Industry |
| Biosafety | 2 |
| Culture Medium | Keratinocyte Basal Medium (KBM) supplemented with Keratinocyte Growth Medium (KGM)-Lonza |
| Subculturing | Enzymatic Dissociation: 1. Remove and discard the culture medium. 2. Rinse the flask three times with 1x PBS solution to remove residual metabolites from cellular metabolism. 3. Add 2.0 to 3.0 mL of trypsin-EDTA solution to the flask. 4. Observe the flask under an inverted microscope until the cell layer becomes individualized and detached (usually between 3 and 7 minutes). NOTE: To prevent cell clumping, do not agitate the flask until trypsin has effectively acted. The flask may be placed at 37ºC (optimum trypsin activity temperature) to optimize the process. If, within the expected time, the cells are individualized but still slightly adherent, the flask can be gently tapped against the palm of the hand or a flat, smooth surface. 5. Add a volume of complete growth medium proportional to the previously added trypsin solution (2.0–3.0 mL). 6. Gently mix the cell suspension with the pipette to ensure even distribution of trypsin and medium, then transfer the suspension to a tube. 7. Remove an aliquot for cell counting using a Neubauer chamber or an automated cell counter. 8. Centrifuge the cell suspension. 9. Subculture: Cultures can be established by centrifugation followed by resuspension at a density of 4–6 × 10³ cells/cm². NOTE: For more details on enzymatic dissociation and cell subculture, refer to Chapter 12 of Culture of Animal Cells, 6th edition, by R. Ian Freshney, published by Alan R. Liss, N.Y., 2010. |
| Subculturing Medium Renewal | Every 2 to 3 days |
| Cryopreservation | 50% FBS +40% KBM + 10% 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.
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 | R. Ian Freshney's book Culture of Animal Cells, 6th edition, published by Alan R. Liss, N.Y., 2010. |
