小鼠雪旺细胞Cell Specification
Schwann cells are neural crest derivatives that ensheathe and myelinate axons of peripheral
nerves [1]. They wrap individually around the shaft of peripheral axons, forming myelin sheath
along segments of the axon. Schwann cells play important roles in the development, function and
regeneration of peripheral nerves. When an axon is dying, the Schwann cells surrounding it aid
in its digestion. This leaves an empty channel formed by successive Schwann cells, through
which a new axon may grow from a severed end. The number of Schwann cells in peripheral
nerve is tightly regulated [2]. Their proliferation in vitro can be stimulated by growth factors
including PDGF, FGF, neuregulin and others [3]. The Schwann cells provide a relatively simple,
well-defined and accessible mammalian model for the study of a number of developmental
questions. It is also of particular clinical importance to understand the biology of Schwann cells,
not only in the context of neuropathies and nerve regeneration, but also because the cells or their
precursor might be especially well suited as implants to facilitate repair in the CNS.
MSC from ScienCell Research Laboratories are isolated from neonate day two C57BL/6 mouse
sciatic nerves. MSC are cryopreserved either at primary or passage one culture and delivered
frozen. Each vial contains >5 x 10^5 cells in 1 ml volume. MSC are characterized by
immunofluorescent method with antibodies to S-100, GFAP and CD90. MSC are negative for
mycoplasma, bacteria, yeast and fungi. MSC are guaranteed to further expend 5 population
小鼠雪旺细胞doublings in the conditions provided by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Schwann Cell Medium (SCM, Cat. No. 1701) for the culturing of MSC
in vitro.
Product Use
MSC are for research use only
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.
. They are not approved for human or animal use, or for
application in in vitro diagnostic procedures.
Shipping
Dry ice.
Reference
[1] Jessen, K. R. and Mirsky, R. (1999) Schwann cells and their precursors emerge as major regulators of nerve
development. Trends Neurosci. 22:402-410.
[2] Syroid, D. E., Maycox, P. R., Burrola, P. G., Liu, N., Wen, D., Lee, K. F., Lemke, G., Kilpatrick, T. J. (1996)
Cell death in the Schwann cell lineage and its regulation by neuregulin. Proc. Natl. Acad. Sci. USA 93:9229-
9234.
[3] Rahmatullah, M., Schroering, A., Rothblum, K., Stahl, R. C., Urban, B and Carey, D. J. (1998) Synergistic
regulation of Schwann cells proliferation by heregulin and forskolin. Mol. Cell. Biol. 18:6245-6252.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
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-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
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-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.
小鼠雪旺细胞4. Place the vial in a 37o
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture
vessels. A seeding density of ≥10,000 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, 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 pipet gently resuspends the contents of the vial.
2
6. 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 lysine coated culture vessels that promote
cell attachment.
is recommended.
7. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
caps if necessary to permit gas exchange.
8. Return the culture vessels to the incubator.
9. 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 typical
oval-shaped cell body, with a prominent nucleus and bipolar extensions, giving an overall
spindle shape; 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 over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks (2 μg/cm2
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 37o
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
T-75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37o
C incubator for 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 37o
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 solution
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 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.
小鼠雪旺细胞7. Count cells and plate cells in a new, poly-L-lysine coated flask with cell density as
recommended.
Caution: Handling animal derived products is potentially biohaza dous. Always wear gloves and safety glasses
when working these materials. Never mouth pipette. We recommend following the universal procedures for handling
products of animal 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).