人神经束膜细胞Cell Specification
Perineurial cells are of mesenchymal origin. They make up the perineurium, which has the
important role of maintaining the integrity of the internal peripheral nerve environment by
creating a physical barrier that, under physiologic condition, limits the entry of biologically
active proteins, infectious agents, and migration of blood-borne cells into the nerve bundles [1].
The perineurial cells are characterized by distinct ultrastructural features, including nonbranching
thin cytoplasmic processes coated by an external lamina and joined at their ends by a
tight junction, few organelles, actin and vimentin filaments, and numerous pinocytotic vesicles
[2]. They initially form a loose, permeable sheath around axons and Schwann cells, which may
recruit them from the surrounding mesenchyme, and from which they are separated by the
extracellular matrix. These cells later undergo a mesenchymal-to-epithelial transition, forming
tight junctions and organizing into the perineurium. The perineurial cells are immunoreactive for
vimentin and epithelial membrane antigen but not for the Schwann cell markers S100 protein [3].
人神经束膜细胞HPNC from ScienCell Research Laboratories are isolated from human spinal nerves. HPNC are
cryopreserved at passage one and delivered frozen. Each vial contains >5 x 10^5 cells in 1 ml
volume. HPNC are characterized by immunofluorescent method with antibodies to Vimentin,
S100, GFAP and CD90. HPNC are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast
and fungi. HPNC are guaranteed to further expand for 15 population doublings in the conditions
provided by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Fibroblast Medium (FM, Cat. No. 2301) for the culturing of HPNC in
vitro.
Product Use
HPNC 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] Salzer, J. L. (1999) Creating barriers: a new role for Schwann cells and desert hedgehog. Neuron 22:627629.
[2] Erlandson, R. A. (1991) The enigmatic perineurial cell and its participation in tumors and in tumor like entities.
Ultrastruct Pathol. 15:335-351.
人神经束膜细胞[3] Ariza, A., Bilbao, J. M. and Rosai, J. (1988) Immunohistochemical detection of epithelial membrane antigen in
normal and perineurial cells and perineurioma. Am J Surg Pathol. 12:678-683.
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-L-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 15 μl of poly-L-lysine stock solution (10
mg/ml, ScienCell cat. no. 0413). 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 a 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 cells are plated in poly-L-lysine coated culture vessels 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 stellate or
spindle-shaped cell morphology, nongranular cytoplasm, and the cell number will be
double after two to three days in culture.
Maintenance of Culture:
人神经束膜细胞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 90% confluent.
2. Prepare poly-L-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 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
for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions
人神经束膜细胞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).