CD1小鼠脑血管周细胞Cell Specification
Brain Vascular Pericytes (BVP) are perivascular cells that are closely associated with the
endothelium of capillaries and other small vessels [1]. BVP, located between endothelial cells
and astrocytes in the brain, communicate with other cells by extending long cytoplasmic
processes which wrap around the capillaries [1, 2]. BVP participate in a variety of processes
including angiogenesis, endothelial cell survival, regulation of capillary blood flow, and
establishment and maintenance of the blood-brain barrier [3, 4]. Pericyte dysregulation has been
linked to several pathological conditions such as hypertension, diabetic retinopathy,
atherosclerosis, multiple sclerosis, Alzheimer’s disease, and tumor angiogenesis [2, 4]. The
unique and diverse functions of BVP make them novel candidates for cell therapy in regenerative
medicine. Cultured primary mouse BVP (MBVP) are a useful in vitro model for understanding
the molecular mechanisms of blood-brain barrier regulation and for studying a wide variety of
central nervous system diseases.
MBVP from ScienCell Research Laboratories are isolated from adult mouse brain. MBVP are
cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
cells in 1 ml
volume. MBVP are characterized by immunofluorescence with antibody specific to Neural/glial
antigen 2 (NG2) and -smooth muscle actin. MBVP are negative for mycoplasma, bacteria,
CD1小鼠脑血管周细胞yeast, and fungi. MBVP are guaranteed to further expand for 5 population doublings under the
conditions provided by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Pericyte Medium (PM, Cat. #1201) for culturing MBVP in vitro.
Product Use
MBVP 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] Dore-Duffy P, Cleary K. (2011) “Morphology and properties of pericytes.” Methods Mol Biol. 686:49-68.
[2] Allt G, Lawrenson JG. (2001) “Pericytes: cell biology and pathology.” Cells Tissues Organs. 169: 1-11.
[3] Daneman R, Zhou L, Kebede A, Barres B. (2010) “Pericytes are required for blood-brain barrier integrity during
embryogenesis.” Natur . 468:562-566.
[4] Kutcher M, Herman I. (2009) “The pericyte: cellular regulator of microvascular blood flow.” Microvasc Res. 77:
235-246.
Instructions for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC water bath
and return the cells to culture as quickly as possible with minimal handling!
Initiating the culture:
1. Prepare a poly-L-lysine coated culture vessel (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, Cat. #0413). Leave the vessel in incubator overnight (minimum one
hour at 37oC incubator).
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-L-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 37oC 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.
5. Remove the cap carefully without touching the interior threads. Gently resuspend and
dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture vessel.
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 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 the best result, do not disturb the culture for at least 16 hours after the culture has
been initiated. Refresh culture medium the next day to remove the 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.
CD1小鼠脑血管周细胞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-L-lysine coated culture vessels (2 μg/cm2
) 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 at 37oC water bath
prior use.
4. Rinse the cells with DPBS.
5. Add 8 ml of DPBS and then 2 ml of T/E solution into flask (in the case of T-75 flask).
Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the
flask at 37oC incubator for 1 to 2 minutes or until cells completely 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 detached) and continue to incubate the flask at 37oC for another 1 to 2 minutes (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.
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 the residual cells.
10. Examine under microscope for a successful cell harvest by looking at the number of cells
being left behind. There should be less than 5%.
Note: Use ScienCell T/E solution that is optimized to minimize cell damages due to over
trypsinization.
CD1小鼠脑血管周细胞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-L-lysine coated culture vessel with cell density
as recommended.
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.