人髓核细胞Cell Specification
Nucleus pulposus is the jelly-like substance in the middle of the intervertebral disc. It functions
to distribute hydraulic pressure in all directions within each disc under compressive loads. The
nucleus pulposus consists of collagen fibrils, proteoglycan aggrecans and nucleus pulposus cells.
The nucleus pulposus cells reside in an environment that has a limited vascular supply and
generate energy through anaerobic glycolysis. The cells synthesize collagen II and X; express
hypoxia-inducible factor-1 [1]; and secrete interleukins-1, -6, and -10 and granulocytemacrophage
colony-stimulating factor in culture [2]. Mechanical stress on nucleus pulposus cells
promotes cell proliferation [3]. The nucleus pulposus cell culture provides an in vitro model for
the study of cellular and molecular events involved in disc degeneration, tissue engineering and
cell therapy for spine disc disorders.
HNPC from ScienCell Research Laboratories are isolated from nucleus pulposus of human
invertibral disc. HNPC are cryopreserved at primary or passage one culture and delivered frozen.
Each vial contains >5 x 105
cells in 1 ml volume. HNPC are characterized by immunofluorescent
method with antibodies to fibronectin and vimentin. HNPC are negative for HIV-1, HBV, HCV,
mycoplasma, bacteria, yeast and fungi. HNPC are guaranteed to further expand for 15 population
人髓核细胞doublings in the condition provided by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use nucleus pulposus cell medium (NPCM, Cat. No. 4801) for the culturing
of HNPC in vitro.
Product Use
HNPC 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]. A Agrawal, A Guttapalli, S Narayan, TJ. Albert, IM. Shapiro, and MV. Risbud (2007) Normoxic stabilization of
HIF-1α drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat
intervertebral disk. Am J Physiol Cell Physiol 293: C621-C631.
[2]. K Torsten, N Thomas, G Christoph, G Tatiana (2005) Human Anulus Fibrosis and Nucleus Pulposus Cells of
the Intervertebral Disc: Effect of Degeneration and Culture System on Cell Phenotype. Spine 30(24):2743-
2748.
[3]. M Takuji, K Mamoru, K Koichi, T Toru, T Tetsuya (1999) Cyclic Mechanical Stretch Stress Increases the
Growth Rate and Collagen Synthesis of Nucleus Pulposus Cells In Vitro. Spine 24(4):315-319.
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 poly-lysine coated flask (2 μg/cm2
, T-75 flask is recommended). Add 10 ml of
sterile water to a T-75 flask and then add 150 μl of poly-lysine stock solution (1 mg/ml,
ScienCell cat. no. 0403). Leave the flask in incubator overnight (minimum one hour at
人髓核细胞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-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 a 1 ml eppendorf pipette gently
resuspend the contents of the vial.
5. Dispense the contents of the vial into the equilibrated, poly-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 vacular smooth muscle cells are plated in poly-lysine coated flasks
that promote cell attachment.
6. Replace the cap or cover of flask, and gently rock the vessel to distribute the cells evenly.
Loosen cap 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 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 poly-lysine coated cell culture flasks (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 (cat. no. 0103) 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-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.
人髓核细胞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).