人羊膜上皮细胞Cell Specification
Human amniotic membrane is composed of an epithelial cell layer, a basement membrane and an
avascular matrix. The amniotic epithelial cells (AEC) are formed from epiblasts on the 8th day
after fertilization. A probable result of their embryonic origin, AEC lack major
histocompatibility complex antigens and have been used for allotranplantation to treat patients
with lysosomal diseases. Studies have shown that AEC have multiple functions such as synthesis
and release of acetylcholine and catecholamine as well as expressing mRNA coding for
dopamine receptors and dopamine transporters [1]. They express neuronal and glial cell marks,
produce basic fibroblast growth factors and hepatocyte growth factors and transform growth
factor-beta [2]. Human AEC has been suggested as an appropriate human cell model for studying
dopamine release and uptake processes, receptor signal transduction and exploring newly
developed drugs acting at these receptors [3].
HAEpiC from ScienCell Research Laboratories are isolated from human amniotic membranes.
HAEpiC are cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
Recommended Medium
cells
in 1 ml volume. HAEpiC are characterized by immunofluorescent method with antibodies to
cytokeratin-18, -19 and vimentin. HAEpiC are negative for HIV-1, HBV, HCV, mycoplasma,
bacteria, yeast and fungi. HAEpiC are guaranteed to further expand for 15 population doublings
人羊膜上皮细胞in the condition provided by ScienCell Research Laboratories.
It is recommended to use Epithelial Cell Medium (EpiCM, Cat. No. 4101) for the culturing of
HAEpiC in vitro.
Product Use
HAEpiC are for research use only
Storage
. It is not approved for human or animal use, or for application
in in vitro diagnostic procedures.
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] Sakuragawa, N., Elwan, M. A., Uchida, S., Fujii, T., Kawashima, K. (2001) Non-neuronal neurotransmitters and
neurotrophic factors in amniotic epithelial cells: expression and function in humans and monkey. Jpn J Pharmacol.
85(1):20-3.
[2] Ishii, T., Ohsugi, K., Nakamura, S., Sato, K., Hashimoto, M., Mikoshiba, K., Sakuragawa, N. (1999) Gene
expression of oligodendrocyte markers in human amniotic epithelial cells using neural cell-type-specific expression
system. Neurosci Lett. 268(3):131-4.
[3] Elwan, M. A., Ishii, T., Sakuragawa, N. (2003) Characterization of the dopamine transporter gene expression and
binding sites in cultured human amniotic epithelial cells. Neurosci Lett. 342(1-2):61-4.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
and return them to culture as quickly as possible with minimal handling!
C waterbath
Set up culture after receiving the order:
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
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.
人羊膜上皮细胞C incubator).
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 37o
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture
vessels. A seeding density of 5,000 cells/cm
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.
2
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 lysine coated flask that promotes cell
attachment and growth.
is recommended.
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 health culture will display polygonal
shaped, sheets of contiguous cells and the cell number will be double after two to three
days in culture.
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 37o
4. Rinse the cells with DPBS.
C
waterbath prior to use.
人羊膜上皮细胞5. Add 7 ml of DPBS first and then 3 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 37o
C incubator for 1 to 3 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 37o
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
C for 1 or 2 minutes more (no
solution 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%.
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.
7. Count cells and plate cells in a new, poly-L-lysine coated flask with cell density as
recommended.
Caution: Handling human derived products is potentially biohaza dous. 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 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).