大鼠神经束膜细胞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 non
branching 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].
PNC from ScienCell Research Laboratories are isolated from Rat spinal nerves. RPNC are
cryopreserved at passage one and delivered frozen. Each vial contains >5 x 10
Recommended Medium
cells in 1 ml
volume. RPNC are characterized by immunofluorescent method with antibodies to Vimentin,
100, GFAP and CD90. RPNC are negative for mycoplasma, bacteria, yeast and fungi. RPNC
are guaranteed to further expand for 5 population doublings in the conditions provided by
ScienCell Research Laboratories.
It is recommended to use Fibroblast Medium (FM, Cat. No. 2301) for the culturing of PNC in
vitro
Product Use
大鼠神经束膜细胞PNC are for research use only
Storage
. They are not approved for human or animal use, or for
application in in vitro diagnostic procedures.
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.
[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 37
and return them to culture as quickly as possible with minimal handling!
C waterbath
Set up culture after receiving the order:
1. Prepare a poly lysine coated flask (2 μg/cm , 75 flask is recommended). Add 10 ml
of sterile water to a T 75 flask and then add 15 μl of poly lysine stock solution (10
mg/ml, ScienCell cat. no. 0413). Leave the flask in incubator overnight (minimum one
hour at 37
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.
C incubator).
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 37
5. Dispense the contents of the vial into the equilibrated, poly lysine coated culture
vessels. A seeding density of 5,000 cells/cm
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 re
suspend the contents of the vial.
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
大鼠神经束膜细胞also important that cells are plated in poly lysine coated flask that promotes cell
attachment and growth.
is recommended.
6. Replace the cap or cover, 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.
Maintenance of Culture:
1. Change the medium to fresh supplemented medium the next morning after establishing a
culture from cryopreserved cells.
2. Change the medium every three days thereafter, until the culture is approximately 70%
confluent.
3. Once the culture reaches 70% confluence, change medium every other 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/cm
3. Warm medium, trypsin/EDTA solution (T/E, cat. no. 0103), trypsin neutralization
solution (TNS, cat. no. 0113), and DPBS (Ca
).
++ and Mg++ free, cat. no. 0303) to room
temperature. We do not recommend warming the reagents and medium at 37
4. Rinse the cells with DPBS.
C
waterbath prior to use.
5. Add 8 ml of DPBS first and then 2 ml of trypsin/EDTA solution into flask (in the case of
75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37 C incubator for 1 2 minutes or until cells are
completely rounded up (monitored with inverted microscope). During incubation, prepare
a 50 ml conical centrifuge tube with 5 ml of fetal bovine serum (FBS, cat. no. 0500);
transfer trypsin/EDTA solution from the flask to the 50 ml centrifuge tube (a few percent
of cells may detached); continue incubate the flask at 37
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
C for 1 or 2 minutes more (no
olution in the flask at this moment); at the end of trypsinization, one hand hold one side
of flask and the other hand gently tap the other side of the flask to detach cells from
attachment; check the flask under inverted microscope to make sure all cells are
detached, add 5 ml of trypsin neutralization solution to the flask and transfer detached
cells to the 50 ml centrifuge tube; add another 5 ml of TNS to harvest the residue cells
大鼠神经束膜细胞and transfer it to the 50 ml centrifuge tube. Examine the flask under inverted microscope
to make sure the cell harvesting is successful by looking at the number of cells left
behind. There should be less than 5%.
6. Centrifuge the 50 ml centrifuge tube (harvested cell suspension) at 1000 rpm (Beckman
Coulter Allegra 6R centrifuge or similar) for 5 min; re suspend cells in growth medium.
7. Count cells and plate cells in a new, poly lysine coated flask with cell density as
recommended.
Caution: Handling animal derived products is potentially bioharzadous. Proper precautions mus 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).