大鼠雪旺细胞Cell Specification
Schwann cells are neural crest derivatives that ensheathe and myelinate axons of peripheral
nerves [1]. Each Schwann cell wrap around the shaft of an individual peripheral axon, 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, leaving an empty channel formed by successive
Schwann cells, through which a new axon may then 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 various growth factors including PDGF, FGF, neuregulin and others [3]. 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 in understanding
the biology of Schwann cells, not only in the context of neuropathies and nerve regeneration, but
also because the cells or their precursor may be especially well suited for implants to facilitate
repair in the CNS.
RSC from ScienCell Research Laboratories are isolated from neonatal ra sciatic nerves. RSC are
cryopreserved either at primary or passage one culture and delivered frozen. Each vial contains
5 x 10 cells in 1 ml volume. RSC are characterized by immunofluorescence with antibodies
specific to S 100, GFAP and CD90. RSC are negative for mycoplasma, bacteria, yeast and
大鼠雪旺细胞fungi. RSC are guaranteed to further culture under the conditions provided by ScienCell
Research Laboratories.
Recommended Medium
It is recommended to use Schwann Cell Medium (SCM, Cat. 1701) for culturing RSC in vitro.
Product Use
RSC are for research use only. They are not approved for human or animal use, or for application
in in vitro diagnostic procedures.
Storage
Upon receiving, directly and immediately transfer the cells from dry ice to liquid nitrogen and
keep the cells in liquid nitrogen until they are needed for experiments.
Shipping
Dry ice.
References
[1] Jessen KR Mirsky R. (1999) “Schwann cells and their precursors emerge as major regulators of nerve
development.” Trends Neurosci. 22: 402 10.
[2] Syroid DE, Maycox PR, Burrola PG, Liu N, Wen D, Lee KF, Lemke G, Kilpatrick TJ. (1996) “Cell death in the
Schwann cell lineage and its regulation by neuregulin.” Proc Natl Acad Sci USA 93: 9229 34.
[3] Rahmatullah M, Schroering A, Rothblum K, Stahl RC, Urban B Carey DJ. (1998) “Synergistic regulation of
Schwann cells proliferation by heregulin and forskolin.” Mol Cell Biol. 18: 6245 52.
Instructions for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37 C water bath
and return the cells to culture as quickly as possible with minimal handling!
Initiating the culture:
1. Prepare a poly lysine coated culture vessel (2 μg/cm , T 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, Cat. #0413). Leave the vessel in a 37 C incubator overnight
(minimum one hour).
2. Prepare complete medium. Decontaminate the external surfaces of medium bottle and
medium supplement tubes with 70% ethanol and transfer them to a sterile field.
Aseptically transfer supplement to the basal medium with a pipette. Rinse the tube with
medium to recover the entire volume.
3. Rinse the poly lysine coated vessel with sterile water twice and then add 15 ml of
complete medium. Leave the vessel in the sterile field and proceed to thaw the
cryopreserved cells.
4. Place the frozen vial in a 37 C water bath. Hold and rotate the vial gently until the
contents completely thaw. Remove the vial from the water bath promptly, wipe it down
with 70% ethanol and transfer it to the sterile field.
大鼠雪旺细胞5. Remove the cap carefully without touching the interior threads. Gently resuspend and
dispense the contents of the vial into the equilibrated, poly lysine coated culture vessel.
A seeding density of ≥10,000 cells/cm 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 residual DMSO in the culture. It is
also important that cells are plated in poly lysine coated culture vessels to promote cell
attachment.
6. Replace the cap or lid and gently rock the vessel to distribute the cells evenly. Loosen cap
if necessary to allow gas exchange.
7. Return the culture vessel to the incubator.
8. For best results, do not disturb the culture for at least 16 hours after the culture has been
initiated. Refresh culture medium the next day to remove residual DMSO and unattached
cells, then every other day thereafter.
Maintaining the culture:
1. Refresh supplemented culture 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% confluency, change medium every other day until the
culture is approximately 90% confluent.
Subculturing:
1. Subculture when the culture reaches 90% confluency or above.
2. Prepare poly lysine coated culture vessels (2 μg/cm ) one day before subculture.
3. Warm complete medium, trypsin/EDTA solution (T/E, Cat. #0103), T/E neutralization
solution (TNS, Cat. #0113), and DPBS (Ca
++
and Mg
++
free, Cat. #0303) to room
temperature. We do not recommend warming reagents and medium in a 37 C water bath
prior to use.
4. Rinse the cells with DPBS.
5. Add 9 ml of DPBS and then 1 ml of T/E solution into flask (in the case of 75 flask).
Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the
flask in a 37 C incubator for 1 minute or until cells start to round up. Use microscope to
monitor the change in cell morphology.
6. During incubation, prepare a 50 ml conical centrifuge tube with 5 ml of fetal bovine
serum (FBS, Cat. #0500).
7. Transfer T/E solution from the flask to the 50 ml centrifuge tube (a few percent of cells
may detach) and continue to incubate the flask at 37 C for another minute (no solution in
the flask at this moment).
8. At the end of incubation, gently tap the side of the flask to dislodge cells from the
surface. Check under microscope to make sure that all cells detach.
大鼠雪旺细胞9. Add 5 ml of TNS solution to the flask and transfer detached cells to the 50 ml centrifuge
tube. Rinse the flask with another 5 ml of TNS to collect residual cells.
10. Examine the flask under microscope for a successful cell harvest by looking at the
number of cells left behind here should be less than 5%.
Note: Use ScienCell T/E solution that is optimized to minimize cell damages due to over
trypsinization.
11. Centrifuge the 50 ml centrifuge tube at 1000 rpm for 5 min. Resuspend cells in culture
medium.
12. Count and plate cells in a new, poly lysine coated culture vessel with
recommended cell density.
Caution: Handling animal derived products is potentially biohazardous. Always wear gloves
and safety glasses when working with 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 WE, Polt S. (1988) “Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues.” J Tissue Cult Methods. 11: 191 9.