人直肠成纤维细胞Cell Specification
The human rectum is located in the lower gastrointestinal tract and functions as a temporary
storage site for fecal material. Rectal fibroblasts are important structural components of the
rectum and are implicated in a number of diseases including ulcerative colitis, Crohn’s disease,
and rectal cancer. Fibroblasts are mesenchymal cells derived from the embryonic mesoderm. The
main functions of fibroblasts are to maintain the structural integrity of connective tissue and to
aid in tissue repair and remodeling [1, 2]. Studies have shown that functional changes to human
rectal fibroblasts (HRecF) can impair wound healing and affect epithelial cell proliferation in
ulcerative colitis patients [3]. In Crohn’s disease, HRecF respond to intestinal inflammation by
proliferating abnormally, which leads to fibrotic scarring. Primary HRecF can be used to study
the pathophysiology of rectal cancer, ulcerative colitis, and Crohn’s disease and to help develop
therapies for these diseases.
HRecF from ScienCell Research Laboratories are isolated from human rectum. HRecF are
cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
cells in 1 ml
volume. HRecF are characterized by immunofluorescence with antibody specific to fibronectin.
HRecF are negative for mycoplasma, bacteria, yeast, and fungi. HRecF are guaranteed to further
expand for 15 population doublings under the conditions provided by ScienCell Research
人直肠成纤维细胞Laboratories.
Recommended Medium
It is recommended to use Fibroblast Medium (FM, Cat. #2301) for culturing HRecF in vitro.
Product Use
HRecF 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.
References
[1] 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.
[2] Conrad GW, Hart GW, Chen Y. (1977) “Differences in vitro between fibroblast-like cells from cornea, heart,
and skin of embryonic chicks.” J Cell Sci. 26: 119-137.
[3] Getliffe K, Martin Ruiz C, Passos JF, von Zglinicki T, Nwokolo C. (2006) “Extended lifespan and long
telomeres in rectal fibroblasts from late-onset ulcerative colitis patients.” Eur J Gastroenterol Hepatol. 18(2): 133-
141.
Instructions 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.
人直肠成纤维细胞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.
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 90-95% confluency.
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.
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, Cat. #0500).
人直肠成纤维细胞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.
Caution: Handling animal derived products is potentially biohazardous. 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.