大鼠皮肤成纤维细胞-成年Cell Specification
Fibroblasts are mesenchymal cells derived from the embryonic mesoderm. They have been
extensively used for a wide range of cellular and molecular studies. This is mainly because they
are one of easiest types of cells to grow in culture, and their durability makes them amenable to a
wide variety of manipulations ranging from studies employing gene transfection to
microinjection. There is good evidence that fibroblasts in different parts of the body are
intrinsically different [1]. Fibroblasts within tissues are exposed to a dynamic mechnical
environment, which influences the structure integrity of both healthy and healing soft tissue.
Fibroblasts secrete a nonrigid extracellular matrix that is rich in type I and/or type III collagen
[2]. In addition, dermal fibroblasts also secrete large quantities of hyaluronan in response to
inflammatory stimuli [3]. During wound healing, dermal fibroblasts switch from a migratory,
repopulating phenotype to a contractile, matrix-reassembling phenotype.
RDF-a from ScienCell Research Laboratories are isolated from adult rat skin. RDF-a are
cryopreserved at primary culture and delivered frozen. Each vial contains >5 x 105
cells in 1 ml
volume. RDF-a are characterized by their spindle morphology and immunofluorescent method
with antibody to fibronectin. RDF-a are negative for mycoplasma, bacteria, yeast and fungi.
大鼠皮肤成纤维细胞-成年RDF-a are guaranteed to further expand for 15 population doublings at the condition provided by
ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Fibroblast Medium (FM, Cat. No. 2301) for the culturing of RDF-a in
vitro.
Product Use
RDF-a 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] Conrad, G. W., Hart, G. W., Chen, Y. (1977) Differences in vitro between fibroblast-like cells from cornea,
heart, and skin of embryonic chicks. J. Cell Sci. 26:119-137.
[2] Gabbiani, G., Rungger-Brandle, E., The fibroblast. In Tissue Repair and Regeneration (L. E. Glynn, ed.), pp 1-
50. Handbook of Inflammation, Vol. 3. Amsterdam, Elsevier, 1981.
[3] Stair S, Carlson KW, Shuster S, Wei ET, Stern R (2002) Mystixin peptides reduce hyaluronan deposition and
edema formation. Eur J Pharmacol 30;450(3):291-6.
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 ordering:
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 15 μl of poly-L-lysine stock solution
(10 mg/ml, ScienCell cat. no. 0413). Leave the flask in incubator overnight (minimum
大鼠皮肤成纤维细胞-成年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 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 1 ml eppendorf pipette
gently re-suspend 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-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 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 flasks (2 μg/cm2
) one day before subculture.
3. Warm medium, trypsin/EDTA solution (T/E, cat. no. 0103), trypsin neutralization
solution (TNS, cat. no. 0113), and DPBS 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 8 ml of DPBS first and then 2 ml of trypsin/EDTA solution into flask (in the case of
大鼠皮肤成纤维细胞-成年T-75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37oC incubator for 1 to 3 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 minutes (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.
7. Count cells and plate cells in a new, poly-L-lysine coated flask with cell density as
recommended.
大鼠皮肤成纤维细胞-成年Caution: Handling animal derived products is potentially biohazardous. Always wear gloves and safety glasses
when working these materials. Never mouth pipette. We recommend following the universal procedures for
handling products of animal origin as the minimum 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).