人心脏成纤维细胞Cell Specification
The cardiac fibroblasts (CF) are the principal cell type of the heart. They provide structural
support for cardiac myocytes and are responsible for extracellular matrix synthesis in the heart
during growth and pathophysiological conditions. CF are an important cellular component of
myocardial responses to injury and the source of paracrine growth factors. Their proliferation and
synthesis of matrix is essential for scar formation at sites of myocardial infarction [1], cardiac
fibrosis, and is often complicated by cardiac hypertrophy [2]. CF also respond to changing
environments in pathophysiological conditions by modulating integrin expression [3]. CF culture
has been widely used as a model to study the cardiac matrix remodeled by physiological
(exercise) and pathological (hypertension) stressors.
HCF from ScienCell Research Laboratories are isolated from human heart. HCF are
cryopreserved at secondary culture and delivered frozen. Each vial contains >5 x 105
cells in 1
ml volume. HCF are characterized by immunofluorescent method with antibody to fibronectin.
人心脏成纤维细胞HCF are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HCF 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 HCF in
vitro.
Product Use
HCF 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] Sabri, A., Short, J., Guo, J., Steinberg, S. F. (2002) Protease-activated receptor-1-mediated DNA synthesis in
cardiac fibroblast is via epidermal growth factor receptor transactivation: distinct PAR-1 signaling pathways in
cardiac fibroblasts and cardiomyocytes. Circ. Res. 91:532-9.
[2] Akiyama-Uchida, Y. et al., (2002) Norepinephrine enhances fibrosis mediated by TGF-beta in cardiac
fibroblasts. Hypertension 40:148-54.
[3] Burgess, M. L., Terracio, L, Hirozane, T., Borg, T. K. (2002) Differential integrin expression by cardiac
fibroblasts from hypertensive and exercise-trained rat hearts. Cardiovasc Pathol 11(2):78-87.
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) and leave
the flask in incubator overnight (minimum one hour at 37o
人心脏成纤维细胞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 fibroblast attachment.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
caps 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. A healthy culture will display stellate or
spindle-shaped cell morphology, nongranular cytoplasm, and the cell number will be
doubled after two to three days in culture.
Maintenance of Culture:
1. Change the medium to fresh supplemented medium the next morning after establishing a
culture from cryopreserved cells. For subsequent subcultures, change medium 48 hours
after establishing the subculture.
2. Change the medium every other day thereafter, until the culture is approximately 50%
confluent.
3. Once the culture reaches 50% confluence, change medium every day until the culture is
approximately 80% confluent.
Subculture:
1. Subculture the cells when they are over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks.
3. Warm medium, trypsin/EDTA solution, trypsin neutralization solution, 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. Incubate cells with 10 ml of trypsin/EDTA solution (in the case of T-75 flask) until 80%
of cells are rounded up (monitored with microscope). Add 10 ml of trypsin neutralization
solution to the digestion immediately and gently rock the culture vessel.
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
6. Harvest and transfer released cells into a 50 ml centrifuge tube. Rinse the flask with
another 10 ml of growth medium to collect the residue cells. Examine the flask under
microscope to make sure the harvesting is successful by looking at the number of cells
left behind. There should be less than 5%.
7. Centrifuge the harvested cell suspension at 1000 rpm for 5 min and resuspend cells in
growth medium.
8. Count cells and plate them in a new, poly-L-lysine coated flask with cell density as
recommended.
人心脏成纤维细胞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).