人口腔角质细胞Cell Specification
Oral keratinocytes act as the major barrier to physical, microbial, and chemical agents that may
cause local cell injury. They are involved in the proinflammatory process through the production
of cytokines either constitutively or after a variety of stimuli [1], implying that they may
potentially participate in controlling oral infections through an inflammatory process involving
different interleukins, such as IL-1ß and IL-18 [2]. Oral keratinocytes express a variety of
differentiation markers, the expression of which is influenced by calcium-induced changes in the
transcription of target genes [3]. Oral keratinocytes share major structural and functional features
with the well-characterized dermal keratinocyte and provide a good model to study basic
keratinocyte biology as well as processes of immortalization and malignant transformation.
HOK from ScienCell Research Laboratories are isolated from human oral mucosa. HOK are
cryopreserved on passage one culture and delivered frozen. Each vial contains >5 x 105
cells in
1 ml volume. HOK are characterized by immunofluorescent method with antibodies to
cytokeratine-8, -18 and -19. HOK are negative for HIV-1, HBV, HCV, mycoplasma, bacteria,
yeast and fungi. HOK are guaranteed to further expand for 10 population doublings in the
condition provided by ScienCell Research Laboratories.
Recommended Medium
人口腔角质细胞It is recommended to use Oral Keratinocyte Medium (OKM, Cat. No. 2611) for the culturing of
HOK in vitro.
Product Use
HOK are for research use only. It is not approved for human or animal use, or for application in
in vitro diagnostic procedures.
Storage
Directly and immediately transfer cells from dry ice to liquid nitrogen upon receiving and keep
the cells in liquid nitrogen until cell culture needed for experiments.
Shipping
Dry ice.
Reference
[1]. Lundqvist, C., Baranov, V., Teglund, S., Hammarstrom, S. and Hammarstrom, M.L. (1994) Cytokine profile
and ultrastructure of intraepithelial gamma delta T cells in chronically inflamed human gingiva suggest a
cytotoxic effector function. J. Immunol. 153:2302-2312.
[2]. M. Rouabhia, G. Ross, N. Page, and J. Chakir (2002) Interleukin-18 and Gamma Interferon Production by Oral
Epithelial Cells in Response to Exposure to Candida albicans or Lipopolysaccharide Stimulation. Infect. Immun.
70:7073-7080.
[3]. Presland, R.B. and Dale, B.A. (2000) Epithelial structural proteins of the skin and oral cavity: function in health
and disease. Crit. Rev. Oral Biol. Med. 11:383-408.
ScienCell
Research Laboratories
TM
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC waterbath
and return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the order:
1. Prepare a poly-L-lysine coated flask (2 μg/cm2
, T-75 flask is recommended). Add 10 ml
of sterile water to a T-75 flask and then add 15μl of poly-L-lysine stock solution (10
mg/ml, ScienCell cat. no. 0413). Leave the flask in incubator overnight (minimum one
hour at 37oC incubator).
2. Prepare complete medium: decontaminate the external surfaces of medium and medium
supplements with 70% ethanol and transfer them to sterile field. Aseptically open each
supplement tube and add them to the basal medium with a pipette. Rinse each tube with
人口腔角质细胞medium to recover the entire volume.
3. Rinse the poly-L-lysine coated flask with sterile water twice and add 20 ml of complete
medium to the flask. Leave the flask in the hood and go to thaw the cells.
4. Place the vial in a 37oC waterbath, hold and rotate the vial gently until the contents are
completely thawed. Remove the vial from the waterbath immediately, wipe it dry, rinse
the vial with 70% ethanol and transfer it to a sterile field. Remove the cap, being careful
not to touch the interior threads with fingers. Using a 1ml eppendorf pipette gently resuspend
the contents of the vial.
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture
vessels. A seeding density of 5,000 cells/cm2
is recommended.
Note: Dilution and centrifugation of cells after thawing are not recommended since these
actions are more harmful to the cells than the effect of DMSO residue in the culture. It is
also important that cells are plated in poly lysine coated flask that promotes cell
attachment and growth.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
cap if necessary to permit gas exchange.
7. Return the culture vessels to the incubator.
8. For best result, do not disturb the culture for at least 16 hours after the culture has been
initiated. Change the growth medium the next day to remove the residual DMSO and
unattached cells, then every other day thereafter.
Maintenance of Culture:
1. Change the medium to fresh supplemented medium the next morning after establishing a
culture from cryopreserved cells.
2. Change the medium every three days thereafter, until the culture is approximately 70%
confluent.
3. Once the culture reaches 70% confluence, change medium every other day until the
culture is approximately 90% confluent.
Subculture:
1. Subculture the cells when they are over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks (2 μg/cm2
).
3. Warm medium, trypsin/EDTA solution (T/E, cat. no. 0103), trypsin neutralization
solution (TNS, cat. no. 0113), and DPBS (Ca++ and Mg++ free, cat. no. 0303) to room
temperature. We do not recommend warming the reagents and medium at 37oC
waterbath prior to use.
4. Rinse the cells with DPBS.
5. Add 5 ml of DPBS first and then 5 ml of trypsin/EDTA solution into flask (in the case of
T-75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37oC incubator for 3 to 5 minutes or until cells are
completely rounded up (monitored with inverted microscope). During incubation, prepare
a 50 ml conical centrifuge tube with 5 ml of fetal bovine serum (FBS, cat. no. 0500);
transfer trypsin/EDTA solution from the flask to the 50 ml centrifuge tube (a few percent
of cells may detached); continue incubate the flask at 37oC for 1 or 2 minutes more (no
solution in the flask at this moment); at the end of trypsinization, one hand hold one side
of flask and the other hand gently tap the other side of the flask to detach cells from
attachment; check the flask under inverted microscope to make sure all cells are
detached, add 5 ml of trypsin neutralization solution to the flask and transfer detached
cells to the 50 ml centrifuge tube; add another 5 ml of TNS to harvest the residue cells
and transfer it to the 50 ml centrifuge tube. Examine the flask under inverted microscope
to make sure the cell harvesting is successful by looking at the number of cells left
behind. There should be less than 5%.
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
6. Centrifuge the 50 ml centrifuge tube (harvested cell suspension) at 1000 rpm (Beckman
Coulter Allegra 6R centrifuge or similar) for 5 min; re-suspend cells in growth medium.
人口腔角质细胞7. Count cells and plate cells in a new, poly-L-lysine coated flask with cell density as
recommended.
Caution: Handling human derived products is potentially biohazardous. Although each cell strain tests negative for
HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions must
be taken to avoid inadvertent exposure. Always wear gloves and safety glasses when working these materials. Never
mouth pipette. We recommend following the universal procedures for handling products of human origin as the
inimum precaution against contamination [1].
[1]. Grizzle, W. E., and Polt, S. S. (1988) Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues. J Tissue Culture Methods. 11(4).