人脉络丛成纤维细胞Cell Specification
Fibroblasts are mesenchymal cells, which they derived from the embryonic mesoderm.
Although cells loosely called fibroblasts have been isolated from many tissues and assumed to be
connective tissue cells, fibroblasts appear to be the least specialized cells. There is good
evidence that fibroblasts in different parts of the body are intrinsically different [1]. Fibroblasts
secrete a nonrigid extracellular matrix that is rich in type I and/or type III collagen [2].
Fibroblasts are the easiest of cells to grow in culture-a feature that has made them a favorite
subject for cell biological studies.
HCPF from ScienCell Research Laboratories are isolated from human choroid pluxus. HCPF are
cryopreserved at primary culture and delivered frozen. Each vial contains >5 x 105
cells in 1 ml
volume. HCPF are characterized by immunofluorescent method with antibody to fibronectin.
人脉络丛成纤维细胞HCPF are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HCPF are
guaranteed to further expand for 15 population doublings in the condition provided by ScienCell
Research Laboratories.
Recommended Medium
It is recommended to use Fibroblast Medium (FM, Cat. No. 2301) for the culturing of HCPF in
vitro.
Product Use
HCPF 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-
人脉络丛成纤维细胞50. Handbook of Inflammation, Vol. 3. Amsterdam, Elsevier, 1981.
ScienCell
Research Laboratories
TM
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!
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 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
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:
人脉络丛成纤维细胞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 37o
C 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
T-75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37o
C 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 37o
C for 1 minutes (no solution in
the flask at this moment); at the end of trypsinisation, 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.
人脉络丛成纤维细胞Caution: Handling human derived products is potentially bioharzadous. Although each cell strain testes negative
for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions
mush 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 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).