小鼠脑膜细胞Cell Specification
Meningeal cells surrounding the brain participate actively in the normal development of the
central nervous system. For example, they play important roles in both stabilizing the
extracellular matrix of the pial surface and by organizing the radial glial scaffold and the
lamination of the cerebellar cortex [1]. Selective pharmacological destruction of the meningeal
cells during a critical ontogenetic period leads to specific malformation of both the cerebella
cortex and dentate gyrus [1]. Grafts of meningeal cells, which are derived from meninges
overlying the cerebral cortex in adult rat spinal cord lesion, promote axonal regrowth [2]. In vitro
study show that meningeal cell chemotactically orients the migration of immature neurons but
not glial cells [3].
MMC from ScienCell Research Laboratories are isolated from C57BL/6 mouse leptomeningi.
MMC are cryopreserved at passage one cultures and delivered frozen. Each vial contains >5 x
105
cells in 1 ml volume. MMC are characterized by immunofluorescent method with antibodies
to fibronectin and negative to GFAP, alpha-smooth muscle actin and Thy 1.1. MMC are negative
for mycoplasma, bacteria, yeast and fungi. MMC are guaranteed to further expand for 5
population doublings in the conditions specified by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Meningeal Cell Medium (MCM, Cat. No. 1401) for the culturing of
MMC in vitro.
Product Use
MMC 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 is needed for experiments.
Shipping
Dry ice.
Reference
[1] Hartmann, D., Sievers, J. Pehlemann, F. W. and Berry, M. (1992) Destruction of meningeal cells over the medial
cerebral hemisphere of newborn hamster prevents the formation of the infrapyramidal blade of the dentate gyrus. J.
Comparative Neurol. 320:33-61.
[2] Franzen, R., Martin, D., Daloze, A., Moonen, G. and Schoenen, J. (1999) Grafts of meningeal fibroblasts in adult
rat spinal cord lesion promote axonal regrowth. Neuroreport 10:1551-1556.
[3] Hartmann, D., Schulze, M. and Sievers, J. (1998) Meningeal cells stimulate and direct the migration of cerebellar
external granule cells in vitro. J. Neurocytol. 27:395-409.
ScienCell
Research Laboratories
TM
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC waterbath
and return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the order:
小鼠脑膜细胞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 37oC 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 37oC 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.
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 flask that promotes cell
attachment and growth.
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-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 37oC
waterbath prior to use.
4. Rinse the cells with DPBS.
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 37oC 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 37oC for 1 or 2 minutes more (no solution
小鼠脑膜细胞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%.
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
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.
小鼠脑膜细胞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 biohazardous. Always wear gloves and safety glasses
when working these materials. Never mouth pipette. We recommend following the universal procedures for
handling products of human and 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).