人子宫肌层微血管内皮细胞Cell Specification
Myometrial microvascular endothelial cells (MMEC) are located in the myometrium between the
endometrium and the serosa of the uterus and exhibit typical cobblestone morphology. During
pregnancy, angiogenesis occurs through MMEC proliferation in order to support the hypertrophy
of the myometrial smooth muscle cell layer [1]. Significant evidence also indicates that
endothelial cell dysfunction in the myometrium may contribute to the development of
preeclampsia [2]. In addition, MMEC proliferation and angiogenesis may play a role in the
formation of leiomyomata [2]. Studies have shown that MMEC express the oxytocin receptor,
which may promote vasodilation in the microvasculature of the myometrium [3]. Human MMEC
are a useful in vitro model to elucidate the mechanisms of angiogenesis and to develop
treatments for female reproductive tract disorders.
HMMEC from ScienCell Research Laboratories are isolated from human uterus. HMMEC are
cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
cells in 1 ml
人子宫肌层微血管内皮细胞volume. HMMEC are characterized by immunofluorescence with antibodies specific to
vWF/Factor VIII and CD31 (PECAM), and by uptake of DiI-Ac-LDL. HMMEC are negative
for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. HMMEC are guaranteed to
further expand for 10 population doublings under the conditions provided by ScienCell Research
Laboratories.
Recommended Medium
It is recommended to use Endothelial Cell Medium (ECM, Cat. #1001) for culturing HMMEC in
vitro.
Product Use
HMMEC 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] Gargett C, Bucak K, Rogers P. (2000) “Isolation, characterization and long-term culture of human myometrial
microvascular endothelial cells.” Hum Reprod. 15(2): 293-301.
[2] Hayman R, Brockelsby J, Kenny L, Baker P. (1999) “Preeclampsia: the endothelium, circulating factor(s) and
vascular endothelial growth factor.” J So Gynecol Investig. 6(1): 3-10.
[3] Weston G, Cann L, Rogers P. (2003) “Myometrial microvascular endothelial cells express oxytocin receptor.”
BJOG. 110(2):149-156.
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 fibronectin-coated culture vessel (2 μg/cm2
, T-75 flask is recommended). Add
5 ml of sterile Dulbecco’s phosphate buffered saline, Ca++
- and Mg++
-free (Cat. #0303) to
a T-75 flask and then add 150 μl of fibronectin stock solution (Cat. #8248). Leave the
vessel in a 37oC incubator overnight.
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 supplement
tube with medium to recover the entire volume.
3. Aspirate fibronectin solution and add 15 ml of complete medium to the culture vessel.
The fibronectin solution can be used twice. 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. Promptly remove the vial from the water bath, wipe it down
with 70% ethanol, and transfer it to the sterile field.
人子宫肌层微血管内皮细胞5. Carefully remove the cap without touching the interior threads. Gently resuspend and
dispense the contents of the vial into the equilibrated, fibronectin-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 fibronectin coated culture vessels to promote cell
attachment.
6. Replace the cap or lid of the culture vessel 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 fibronectin-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 in a 37oC water bath
prior to use.
4. Rinse the cells with DPBS.
5. Add 10 ml of DPBS and then 2 ml of T/E solution into flask (in the case of a T-75 flask).
Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the
flask in a 37
oC incubator for 1 to 2 minutes or until cells completely round up. Use a
microscope to monitor the change in cell morphology.
人子宫肌层微血管内皮细胞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 small percent of cells
may detach) and continue to incubate the flask at 37oC for another 1 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 a 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 the flask under a 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.
11. Centrifuge the 50 ml centrifuge tube at 1000 rpm for 5 minutes. Resuspend cells in
culture medium.
12. Count and plate cells in a new fibronectin-coated culture vessel with the recommended
cell density.
Caution: Handling human derived products is potentially biohazardous. Although each cell
strain tests negative for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100%
accurate, therefore, proper precautions must be taken to avoid inadvertent exposure. 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.