人心肌细胞Cell Specification
The cardiac myocyte is the most physically energetic cell in the body. Its contraction is myogenic,
i.e. it is independent of nervous stimulation. All cardiac myocyte are capable of spontaneous
rhythmic depolarization and repolarization of their membrane. Cardiac myocytes occupy as much as
75% of cardiac mass but constitute only about one third of the total cell number in the heart. They are
highly specialized high-oxygen-content cells and house a large number of mitochondria [1].
Differentiated cardiac myocytes have little capacity to proliferate and show the hypertrophic growth
in response to alpha1-adrenergic stimuli via the Ras/MEK pathway [2]. Cardiac myocyte
hypertrophy and apoptosis have been implicated in the loss of contractile function during heart
failure. Cardiac myocytes have a complex network of signals that regulates their essential role in the
rhythmic pumping of the heart [3]. This network is an appealing model system in which to study the
basic principles of cellular signaling mechanisms leading to cardiac myocyte death.
HCM from ScienCell Research Laboratories are isolated from human heart (ventricle). HCM are
cryopreserved immediately after purification and delivered frozen. Each vial contains >5 x 105
cells
in 1 ml volume. HCM are characterized by immunofluorescent method with antibodies to myosin.
人心肌细胞HCM are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HCM are
guaranteed to further culture at the conditions provided by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Cardiac Myocyte Medium (CMM, Cat. No. 6201) for the culturing of
HCM in vitro.
Product Use
HCM 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] Bodyak, N., Kang, P. M., Hiromura, M., Sulijoadikusumo, I., Horikoshi, N., Khrapko, K. and Usheva, A. (2002)
Gene expression profiling of the aging mouse cardiac myocytes. Nucleic Acids Research 30(17):3788-3794.
[2] Tamamori-Adachi, M., Ito, H., Nobori, K., Hayashida, K., Kawauchi, J., Adachi, S., Ikeda, M. A. and Kitajima,
S. (2002) Expression of cyclin D1 and CDK4 causes hypertrophic growth of cardiomyocytes in culture: a
possible implication for cardiac hypertrophy. Biochem Biophys Res Commun 296(2):274-80.
[3] Sambrano, G.R., Fraser, I., Han, H., Ni, Y., OConnell, T., Yan, Z. and Stull, J. T. (2002) Navigating the
signaling network in mouse cardiac myocytes. Nature 420(6916):712-4.
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:
人心肌细胞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
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 7,500 cells/cm2
is recommended.
Note: Dilution and centrifugation of cells after thawing are not recommended since these
actions are more harmful to cells than the effect of DMSO residue in the culture. It is
also important that HCM are plated in poly-l-lysine-coated culture vessels that promote
myocytes attachment and growth.
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 spindle
shaped, usually in a homogeneous bundle or sheet of cells rather than scattered single
cells 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 90% confluent.
Subculture:
1. Subculture the cells when they are over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flask (2 μg/cm2
).
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.
Caution: Handling human derived products is potentially bioharzadous. Although each cell strain testes negative
人心肌细胞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).