大鼠肺成纤维细胞Cell Specification
The most abundant cell type in the lung interstitium is the fibroblast. These fibroblasts resemble
ordinary fibroblasts, but also possess some distinguishing features such as long, branching
processes and gap junctions. Pulmonary fibroblasts function to produce type III collagen, elastin,
and proteoglycans of the extracellular matrix of the alveolar septa. They play an important role in
the repair and remodeling processes following tissue damage. The controlled accumulation of
fibroblasts to sites of inflammation is crucial to effective tissue repair after injury [1]. An
inadequate or an excessive accumulation of fibroblasts can result in abnormal tissue function.
For example, excess proliferation of fibroblasts contributes to adventitial thickening observed
during the development of hypoxia-induced pulmonary hypertension [2].
RPF from ScienCell Research Laboratories are isolated from postnatal day 2 rat lung. RPF are
cryopreserved at P0 and delivered frozen. Each vial contains >5 x 105
cells in 1 ml volume. RPF
are characterized by fibroblast morphology. RPF are negative for mycoplasma, bacteria, yeast,
and fungi. HPF are guaranteed to further expand for 5 population doublings under the conditions
provided by ScienCell Research Laboratories.
大鼠肺成纤维细胞Recommended Medium
It is recommended to use Fibroblast Medium (FM, Cat. #2301) for culturing RPF in vitro.
Product Use
RPF 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] Kuwano K, Hagimoto N, Hara N. (2001) “Molecular mechanisms of pulmonary fibrosis and current treatment.”
Curr Mol Med. 1: 551-73
[2] Das M, Dempsey EC, Reeves JT, Stenmark KR. (2002) “Selective expansion of fibroblast subpopulations from
pulmonary artery adventitia in response to hypoxia.” Am J Physiol Lung Cell Mol Physiol. 282: L976-86.
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 a 37oC incubator overnight (or for a
minimum of one hour).
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 supplement
tube with medium to recover the entire volume.
3. Rinse the poly-L-lysine-coated vessel twice with sterile water 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 37
oC water bath. Hold and rotate the vial gently until the
contents completely thaw. Promptly remove the vial from the water bath, wipe it down
with 70% ethanol, and transfer it to the sterile field.
5. Carefully remove the cap without touching the interior threads. Gently resuspend and
大鼠肺成纤维细胞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 of the culture vessel 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 best results, do not disturb the culture for at least 16 hours after the culture has been
initiated. Refresh culture medium the next day to remove 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 in a 37oC water bath
prior to 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 a T-75 flask).
Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the
flask in a 37oC incubator for 1 to 2 minutes or until cells completely round up. Use a
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).
7. Transfer T/E solution from the flask to the 50 ml centrifuge tube (a small percent of cells
may detach) 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 a 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 the flask under a 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 minutes. Resuspend cells in
culture medium.
12. Count and plate cells in a new poly-L-lysine-coated culture vessel with the recommended
cell density.
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.