人肾小球内皮细胞Cell Specification
Renal glomerular endothelial cells (GEC) are a specialized microvascular cell type involved in
the regulation of glomerular ultrafiltration. They form the inner part of the filtration barrier and
are involved in pathophysiological processes in the glomerulum [1]. They constitutively
synthesize bio-active molecules, and this basal activity can be chronically augmented by various
inflammatory and thrombotic agents [2]. GEC injury exerts significant influences on the
progression and repair process of glomerular disease. When the glomerular lesion is severe,
angiogenesis is prevented due to endothelial cell injury, with subsequent sclerosis taking place in
the impaired region [3]. These glomerular endothelial cell injuries inevitably affect mesangial
and epithelial cells and presumably modify the progression of renal disease by reciprocally
interacting with them [4]. Because of difficulties associated with the culture, cloning and
propagation, the biological properties of these cells remain largely unknown.
HRGEC from ScienCell Research Laboratories are isolated from human kidney. HRGEC are
cryopreserved immediately after purification and delivered frozen. Each vial contains >5 x 10^5
cells in 1 ml volume. HRGEC are characterized by immunofluorescent method with antibodies
to vWF/Factor VIII and CD31 (P-CAM), and by the formation of microtublar structure in vitro.
人肾小球内皮细胞HRGEC are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HRGEC
are guaranteed to further culture at the conditions specified by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Endothelial Cell Medium (ECM, Cat. No. 1001) for the culturing of
HRGEC in vitro.
Product Use
HRGEC are for research use only. They are not approved for human or animal use, or for
application in in vitro diagnostic procedures.
Storage
Transfer cells directly and immediately 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] Nangaku, M., Shankland, S. J., Couser, W. G. and Johnson, R. J. (1998) A new model of renal microvascular
injury. Curr Opin Nephrol Hypertens 7(4):457-62.
[2] Kester, M., Nowinski, R. J., Holthofer, H., Marsden, P. A. and Dunn, M. J. (1994) Characterization of plateletactivating
factor synthesis in glomerular endothelial cell lines. Kidney Int 46(5):1404-12.
[3] Lee, L. K., Meyer, T. W., Pollock, A. S. and Lovett, D. H. (1995) Endothelial cell injury initiates glomerular
sclerosis in the rat remnant kidney. J Clin Invest 96(2):953-64.
[4] Yamanaka, N. and Shimizu, A. (1999) Role of glomerular endothelial damage in progressive renal disease.
人肾小球内皮细胞Kidney Blood Press Res 22(1-2):13-20.
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 order:
1. Prepare a fibronectin coated flask (2 μg/cm2
, T-75 flask is recommended). Add 10 ml of
sterile Dulbecco’s phosphate buffered saline (DPBS) to a T-75 flask and then add 150 μl
of fibronectin stock solution (1 mg/ml, Sigma cat. no. F1141). Leave the flask in
incubator overnight.
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. Aspirate fibronectin solution and add 20 ml of complete medium to the flask. Leave the
flask in the hood and go to thaw the cells. The fibronectin solution can be used twice.
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 a 1 ml eppendorf pipette gently
resuspend the contents of the vial.
5. Dispense the contents of the vial into the equilibrated, fibronectin-coated flask. 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 endothelial cells are plated in fibronectin coated flask that promotes
cell attachment.
6. Replace the cap or cover of flask, and gently rock the flask to distribute the cells evenly.
人肾小球内皮细胞Loosen cap if necessary to permit gas exchange.
7. Return the culture vessels to the incubator.
8. For best results, 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
cobblestone or spindle shaped morphology, nongranular cytoplasm and the cell number
will be double 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 fibronectin coated flasks (2 μg/cm2
) one day before subculture.
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, fibronectin-coated flask with cell density as
recommended.
人肾小球内皮细胞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).