人脑膜细胞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. Selective pharmacological destruction of the meningeal cells
during a critical ontogenetic period leads to specific malformation of both the cerbella cortex and
dentate gyrus [1]. Grafts of meningeal cells, which derived form meninges overlying the cerebral
cortex, in adult rat spinal cord lesion promote axonal regrowth [2]. In vitro study shows that
meningeal cell chemotactically orient the migration of immature neurons [3].
HMC from ScienCell Research Laboratories are isolated from human leptomeningi. HMC are
cryopreserved at primary culture and delivered frozen. Each vial contains >5 x 105
cells in 1 ml
volume. HMC are characterized by immunofluorescent method with antibodies to fibronectin
and negative to GFAP, α-smooth muscle actin and Thy 1.1. HMC are negative for HIV-1, HBV,
HCV, mycoplasma, bacteria, yeast and fungi. HMC are guaranteed to further expand for 15
population doublings at the condition provided by ScienCell Research Laboratories.
人脑膜细胞Recommended Medium
It is recommended to use Meningeal Cell Medium (MCM, Cat. No. 1401) for the culturing of
HMC in vitro.
Product Use
HMC 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 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.
人脑膜细胞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 ordering:
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 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 meningeal cells are plated in poly-L-lysine coated culture vessels that
promote cell attachment.
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. 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:
人脑膜细胞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 over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks.
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
人脑膜细胞for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions
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).