人肝细胞Cell Specification
The liver fulfills many vital processes in mammals. It is the central organ of energy metabolism,
biotransformation of xenobiotics, and synthesis of plasma proteins under physiological and
pathophysiological conditions [1]. Primary culture of human hepatocyte appears to be a suitable
experimental model for the study of liver specific function, have been and still is an important
tool. Propagation of human hepatocytes for cell transplantation, gene therapy, and culture of
hepatocyte in bioartificial liver support systems is now under investigation [2]. In appropriate
culture condition, cultured human hepatocytes proliferate and maintain differentiated hepatocyte
function such as the synthesis of serum proteins [3].
HH from ScienCell Research Laboratories are isolated from human liver. HH are cryopreserved
immediately after purification and delivered frozen. Each vial contains >1 x 106
cells in 1 ml
volume. HH are characterized by immunofluorescent method with antibodies to albumin,
cytokeratin-18 and vimentin. HH are negative for HIV-1, HBV, HCV, mycoplasma, bacteria,
yeast and fungi. HH are guaranteed to further culture in the condition provided by ScienCell
Research Laboratories.
人肝细胞Recommended Medium
It is recommended to use Hepatocyte Medium (HM, Cat. No. 5201) for the culturing of HH in
vitro.
Product Use
HH 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 is needed for experiments.
Shipping
Dry ice.
Reference
[1] Runge, D., Michalopoulos, G. K., Strom, S. C. and Runge, D. M. (2000) Recent advances in human hepatocyte
culture systems. Biochem. Biophysi. Res. Comm. 274:1-3.
[2] Chen, H. L., Wu, H. L., Fon, C. C., Chen, P. J., Lai, M. Y. and Chen, D. S. (1998) Long-term culture of
hepatocytes from human adults. J. Biomed. Sci. 5:435-440.
[3] Okamoto, M., Ishida, Y., Keogh, A. and Strain, A. (1998) Evaluation of the function of primary human
hepatocytes co-cultured with the human hepatic stellate cell (HSC) line LI90. Int. J. Artif. Organs 21:353-359.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
人肝细胞C waterbath
and return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the ordering:
1. 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, and
transfer it to a sterile field. Rinse the vial with 70% ethanol, and then wipe to remove
excess. 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.
2. 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 the cells than the effect of DMSO residue in the culture. It is
also important that HH are plated in poly-L-lysine coated culture vessels that promote
hepatocyte attachment and growth.
3. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
caps if necessary to permit gas exchange.
4. Return the culture vessels to the incubator.
5. 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 health culture will display polygonal
shaped, sheets of contiguous 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 80% confluent.
Subculture:
1. Subculture the cells when they are 80% 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 3 ml of trypsin/EDTA solution (in the case of T-25 flask) until 80% of
cells are rounded up (monitored with microscope). Add 3 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 15 ml centrifuge tube. Rinse the flask with
another 3 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
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.
人肝细胞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).
6.