人牙龈成纤维细胞Cell Specification
Fibroblasts are mesenchymal cells with many vital functions during development and in adult
organisms. They are responsible for much of the synthesis of extracellular matrix in connective
tissues and play major roles in wound healing. Many diseases are associated with fibroblasts,
either because fibroblasts are implicated in their etiology or because of the fibrosis that
accompanies damage to other cell types in tissues. Fibroblasts are among the most accessible
normal mammalian cell types and still the most amenable to culture in vitro. Gingival fibroblasts
are the major constituents of gingival tissue and play a key role in their health maintenance [1].
Human gingival fibroblasts (HGF) express a wide surface molecular panel including mainly
CD9, CD26, CD55, CD59, CD63, CD71, CD86 CD95, CD99 and CD117 [2]. They were also
demonstrated to express mRNAs for protease-activated receptor-1 (PAR-1) and PAR-3 [3].
HGF from ScienCell Research Laboratories are isolated from human gingiva. HGF are
cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
cells in 1 ml
volume. HGF are characterized by their spindle morphology and immunofluorescent method
with antibody to fibronectin. HGF are negative for HIV-1, HBV, HCV, mycoplasma, bacteria,
yeast and fungi. HGF are guaranteed to further expand for 15 population doublings at the
condition provided by ScienCell Research Laboratories.
人牙龈成纤维细胞Recommended Medium
It is recommended to use Fibroblast Medium (FM, Cat. No. 2301) for the culturing of HGF in
vitro.
Product Use
HGF 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] Poggi, P., Rodriguez y Baena, R., Rizzo, S., Rota, M. T. (2003) Mouthrinses with alcohol: cytotoxic effects on
human gingival fibroblasts in vitro. J Periodontol. 74(5):623-9.
[2] Di Domenico, G., Del Vecchio, L., Postiglione, L., Ramaglia, L. (2003) Immunophenotypic analysis of human
gingival fibroblasts and its regulation by Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).
Minerva Stomatol. 52(3):81-7, 87-91.
[3] Tanaka, N., Morita, T., Nezu, A., Tanimura, A., Mizoguchi, I., Tojyo, Y. (2003) Thrombin-induced Ca2+
mobilization in human gingival fibroblasts is mediated by protease-activated receptor-1 (PAR-1). Life Sci.
73(3):301-10.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
C waterbath
and return them to culture as quickly as possible with minimal handling!
人牙龈成纤维细胞Set up culture after receiving the ordering:
1. Prepare a poly-L-lysine coated flask (2 μg/cm2
, T-75 flask is recommended) and leave
the flask in incubator overnight (minimum one hour at 37o
C 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
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 37o
C 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 1 ml 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 fibroblasts are plated in poly-L-lysine coated culture vessels that
promote cell attachment.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
caps if necessary to permit gas exchange.
7. Return the culture vessels to the incubator.
人牙龈成纤维细胞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. A healthy culture will display stellate or
spindle-shaped cell morphology, nongranular cytoplasm, and the cell number will be
doubled after two to three days in culture.
Maintenance of Culture:
1. Change the medium to fresh supplemented medium the next morning after establishing a
culture from cryopreserved cells. For subsequent subcultures, change medium 48 hours
after establishing the subculture.
2. Change the medium every other day thereafter, until the culture is approximately 50%
confluent.
3. Once the culture reaches 50% confluence, change medium every day until the culture is
approximately 80% confluent.
Subculture:
1. Subculture the cells when they are over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks.
3. Warm medirature. 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).