人肾上腺成纤维细胞Cell Specification
The adrenal gland plays an essential role in regulating homeostasis in the body through the
secretion of corticosteroid and androgen hormones. Fibroblasts are mesenchymal cells derived
from the embryonic mesoderm. They have been extensively used for a wide range of cellular and
molecular studies, as they are one of the easiest types of cells to grow in culture. Their durability
makes them amenable to a variety of manipulations ranging from gene transfection to
microinjection [1]. Fibroblasts secrete a non-rigid extracellular matrix that is rich in type I and/or
type III collagen [2]. They are responsible for much of the synthesis of extracellular matrix in
connective tissues and play a central role 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. For example, the development of bowel
stenosis in Crohn’s disease patients is caused by extreme fibroblast proliferation and
extracellular matrix expansion [3].
HAdF from ScienCell Research Laboratories are isolated from human adrenal tissue. HAdF are
cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
cells in 1 ml
volume. HAdF are characterized by their spindle morphology and immunofluorescence with
antibody specific to fibronectin. HAdF are negative for HIV-1, HBV, HCV, mycoplasma,
bacteria, yeast and fungi. HAdF are guaranteed to further expand for 15 population doublings
under the conditions provided by ScienCell Research Laboratories.
人肾上腺成纤维细胞Recommended Medium
It is recommended to use Fibroblast Medium (FM, Cat. #2301) for the culturing of HAdF in
vitro.
Product Use
HAdF are for research use only. They are not approved for human or animal use, or for
application in in vitro diagnostic procedures.
Storage
Upon receiving, directly and immediately transfer the cells from dry ice to liquid nitrogen and
keep the cells in liquid nitrogen until they are needed for experiments.
Shipping
Dry ice.
Reference
[1] Conrad GW, Hart GW, Chen Y. (1977) “Differences in vitro between fibroblast-like cells from cornea, heart,
and skin of embryonic chicks.” Cell Sci. 26: 119-37.
[2] Gabbiani G, Rungger-Brandle E. (1981) “The fibroblast.” In Glynn LE, Handbook of Inflammation, Vol. 3:
Tissue Repair and Regeneration (pp. 1-50) Amsterdam: Elsevier.
[3] Luna J, Masamunt MC, Rickmann M, Mora R, Espana C, Delgado S, Llach, J, Vaquero E, Sans M. (2011)
“Rocotrienols have potent antifibrogenic effects in human intestinal fibroblasts.” Inflamm Bowel Dis. 17: 732-41.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC water bath
and return the cells to culture as quickly as possible with minimal handling!
Initiating the culture:
1. Prepare a poly-L-lysine coated culture vessel (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, Cat. #0413). Leave the vessel in incubator overnight (minimum one
hour at 37oC incubator).
2. Prepare complete medium. Decontaminate the external surfaces of medium bottle and
medium supplement tubes with 70% ethanol and transfer them to a sterile field.
Aseptically transfer supplement to the basal medium with a pipette. Rinse the tube with
medium to recover the entire volume.
人肾上腺成纤维细胞3. Rinse the poly-L-lysine coated vessel with sterile water twice and then add 15 ml of
complete medium. Leave the vessel in the sterile field and proceed to thaw the
cryopreserved cells.
4. Place the frozen vial in a 37oC water bath. Hold and rotate the vial gently until the
contents completely thaw. Remove the vial from the water bath promptly, wipe it down
with 70% ethanol and transfer it to the sterile field.
5. Remove the cap carefully without touching the interior threads. Gently resuspend and
dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture vessel.
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 residual DMSO in the culture. It is
also important that cells are plated in poly lysine coated culture vessels to promote cell
attachment.
6. Replace the cap or lid, and gently rock the vessel to distribute the cells evenly. Loosen
cap if necessary to allow gas exchange.
7. Return the culture vessel to the incubator.
8. For the best result, do not disturb the culture for at least 16 hours after the culture has
been initiated. Refresh culture medium the next day to remove the residual DMSO and
unattached cells, then every other day thereafter.
Maintaining the culture:
1. Refresh supplemented culture 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% confluency, change medium every other day until the
culture is approximately 90% confluent.
Subculturing:
1. Subculture when the culture reaches 90% confluency or above.
2. Prepare poly-L-lysine coated culture vessels (2 μg/cm2
) one day before subculture.
3. Warm complete medium, trypsin/EDTA solution (T/E, Cat. #0103), T/E neutralization
solution (TNS, Cat. #0113), and DPBS (Ca++ and Mg++ free, Cat. #0303) to room
temperature. We do not recommend warming reagents and medium at 37oC water bath
prior use.
4. Rinse the cells with DPBS.
人肾上腺成纤维细胞5. Add 8 ml of DPBS and then 2 ml of T/E solution into flask (in the case of T-75 flask).
Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the
flask at 37oC incubator for 1 to 2 minutes or until cells completely round up. Use
microscope to monitor the change in cell morphology.
6. During incubation, prepare a 50 ml conical centrifuge tube with 5 ml of fetal bovine
serum (FBS, Ca. #0500).
7. Transfer T/E solution from the flask to the 50 ml centrifuge tube (a few percent of cells
may detached) and continue to incubate the flask at 37oC for another 1 to 2 minutes (no
solution in the flask at this moment).
8. At the end of incubation, gently tap the side of the flask to dislodge cells from the
surface. Check under microscope to make sure that all cells detach.
9. Add 5 ml of TNS solution to the flask and transfer detached cells to the 50 ml centrifuge
tube. Rinse the flask with another 5 ml of TNS to collect the residual cells.
10. Examine under microscope for a successful cell harvest by looking at the number of cells
being left behind. There should be less than 5%.
Note: Use ScienCell T/E solution that is optimized to minimize cell damages due to over
trypsinization.
11. Centrifuge the 50 ml centrifuge tube at 1000 rpm for 5 min. Resuspend cells in culture
medium.
12. Count and plate cells in a new, poly-L-lysine coated culture vessel with cell density
as recommended.
人肾上腺成纤维细胞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 with 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 WE, Polt S. (1988) “Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues.” J Tissue Cult Methods. 11: 191-9.